Lighting and DOF ?s

Starting out in microscopy? Post images and ask questions relating to the microscope and get answers from our more advanced users on the subject.

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jmarkus
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Re: Lighting and DOF ?s

Post by jmarkus »

rjlittlefield wrote:
Wed Jan 29, 2025 12:19 am
Thanks for the picture of the objectives.

The constant notation 160/0.17 describes "finite" objectives (no tube lens required) that are designed to look through cover glass that is 0.17 mm thick. Cover glass doesn't really matter for the 10X and 4X objectives, but the 40X has to have it or the image quality will suffer badly.

The notations 100/1.25, 40/0.65, 10/0.25, and 4/0.10 all describe magnification/NA. So, your 40X objective is NA 0.65, and that will have DOF of only about 0.001 mm.

The 100X objective is "oil immersion" and must be used with oil or the image quality will be trash. The oil layer between the objective and cover glass is essentially another refracting element of the lens.

--Rik
Rik,
I guess that cover glass is like the internal UV filter in a super-telephoto lens - a part of the optical formula. I'm a bit concerned about that DOF of 0.001 mm. Yesterday I read about all the mentioned (by Chris S.) stepper hardware (+DYI & others), and decided the WeMicro was the best fit for the microscope, because it could easily also be used with my existing Nikon focusing rail, and the Nikon PB-4 bellows for macro. I purchased one, and it is on the way. I suppose I could decrease the step increment by replacing the Leica BF200 fine adjustment knob with a larger (> 5cm) one - or am I not thinking about this right? Anyway, I don't know if it is capable of doing 1/1000th of a mm slices, but maybe it doesn't have to? I do have the oil for the 100x objective. I assume a cover glass is used for that, and the oil creates a surface tension droplet between the front element of the objective, and the cover glass? However, I just started looking at how to collect my own critters to photograph, and I got some synthetic Balsam from Canada. Was there some balsam kerfuffle? I would think Canada would be swimming in the stuff, but you can't buy it anymore? What happened?
Jim

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Re: Lighting and DOF ?s

Post by rjlittlefield »

jmarkus wrote:
Wed Jan 29, 2025 8:57 am
I suppose I could decrease the step increment by replacing the Leica BF200 fine adjustment knob with a larger (> 5cm) one - or am I not thinking about this right?
Background info: most microscopes used for focus stacking have coaxial focusing knobs, with coarse and fine knobs on the same axis, internally geared together so that you can traverse the whole coarse range by making many turns of the fine focus knob. For example it's typical to have about 25 mm of total movement, with 0.1-0.2 mm per turn of the fine focus knob. With those systems, moving by 0.001 mm steps is trivial because that represents 1/100 or 1/200 turn of the focus knob -- easily within reach of a stepper motor.

But the Leica BF seems to have separate mechanisms for coarse and fine focus (manual HERE). I cannot find a specification for how much range the fine focus knob has, or how much movement per rotation. If it has enough range and small enough movement per rotation, then all you need is some mechanism for firmly gripping it with the MicroMate. That may indeed involve installing a bigger knob, or a different shaft coupler. It might also involve raising the microscope, since that fine focus knob looks pretty close to the base.

One additional thing to watch out for... Those coaxial designs that I mentioned have clutches, so nothing gets damaged if the fine focus knob is turned even after the stage hits a hard stop. I do not know if the Leica BF has something similar on its fine focus knob. If the knob simply runs to a hard stop, then you'll have to be careful to somehow avoid damage from over-driving it with the stepper motor. A simple flexible coupling or clutch might be the easiest approach. The first time I coupled a stepper motor to a fine focus knob, I did the coupling with tapered press fit of some vinyl tubing, see HERE. That looks a bit silly, but it was simple to do and worked fine.
I do have the oil for the 100x objective. I assume a cover glass is used for that, and the oil creates a surface tension droplet between the front element of the objective, and the cover glass?
Correct.

Fair warning: oil is frustrating to work with because it's messy and viscous. Once you've oiled the 100, you can't just drop back to the 40 because the remaining oil will then mess up the image and possibly get into the objective. You have to clean up to drop back.

--Rik

iconoclastica
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Re: Lighting and DOF ?s

Post by iconoclastica »

jmarkus wrote:
Wed Jan 29, 2025 8:57 am
I do have the oil for the 100x objective. I assume a cover glass is used for that, and the oil creates a surface tension droplet between the front element of the objective, and the cover glass?
Even though I have had a 100x from the start, I has been years before I bothered to use it. Well, of course I did try it the first day or so, but without being properly familiar with the microscope and how to handle it, that magnification offers less than satisfactory results. But once you know your microscopy well, then it is very rewarding to make pictures of the smallest what light can see.
--- felix filicis ---

jmarkus
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Re: Lighting and DOF ?s

Post by jmarkus »

rjlittlefield wrote:
Wed Jan 29, 2025 10:39 am
jmarkus wrote:
Wed Jan 29, 2025 8:57 am
I suppose I could decrease the step increment by replacing the Leica BF200 fine adjustment knob with a larger (> 5cm) one - or am I not thinking about this right?
Background info: most microscopes used for focus stacking have coaxial focusing knobs, with coarse and fine knobs on the same axis, internally geared together so that you can traverse the whole coarse range by making many turns of the fine focus knob. For example it's typical to have about 25 mm of total movement, with 0.1-0.2 mm per turn of the fine focus knob. With those systems, moving by 0.001 mm steps is trivial because that represents 1/100 or 1/200 turn of the focus knob -- easily within reach of a stepper motor.

But the Leica BF seems to have separate mechanisms for coarse and fine focus (manual HERE). I cannot find a specification for how much range the fine focus knob has, or how much movement per rotation. If it has enough range and small enough movement per rotation, then all you need is some mechanism for firmly gripping it with the MicroMate. That may indeed involve installing a bigger knob, or a different shaft coupler. It might also involve raising the microscope, since that fine focus knob looks pretty close to the base.

One additional thing to watch out for... Those coaxial designs that I mentioned have clutches, so nothing gets damaged if the fine focus knob is turned even after the stage hits a hard stop. I do not know if the Leica BF has something similar on its fine focus knob. If the knob simply runs to a hard stop, then you'll have to be careful to somehow avoid damage from over-driving it with the stepper motor. A simple flexible coupling or clutch might be the easiest approach. The first time I coupled a stepper motor to a fine focus knob, I did the coupling with tapered press fit of some vinyl tubing, see HERE. That looks a bit silly, but it was simple to do and worked fine.
I do have the oil for the 100x objective. I assume a cover glass is used for that, and the oil creates a surface tension droplet between the front element of the objective, and the cover glass?
Correct.

Fair warning: oil is frustrating to work with because it's messy and viscous. Once you've oiled the 100, you can't just drop back to the 40 because the remaining oil will then mess up the image and possibly get into the objective. You have to clean up to drop back.

--Rik
Rik,
I get the impression that this was a biology student scope. The range I measured is 14mm (it has an adjustable stage stop). It takes five rotations of the fine adjustment knob to move 1mm - so 0.2mm per rotation. The WeMicro focus attachment looks like those medical halos to hold your neck straight after surgery. Four nylon screws grasp the fine adjustment knob with alignment seeming to be the most critical part of hooking it up. Your rubber stoppers and flex hose (that is what it looks like) is an ingenuous solution.

I am pretty sure this microscope was manufactured in 1996. The objectives look just like the E1, and E2 Leica objectives, but no "E" even though the Leica name brand is engraved and painted. I'm still chuffed about getting 3 objectives, an eyepiece, and illumination with a 10,000 hour life - all working for $49.95. Is it the best, no way. It certainly is a nice entry into this area of photography. Could be dangerous for my wallet, but I am having fun.
Jim
Last edited by jmarkus on Thu Jan 30, 2025 7:29 am, edited 1 time in total.

jmarkus
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Re: Lighting and DOF ?s

Post by jmarkus »

iconoclastica wrote:
Wed Jan 29, 2025 12:01 pm
jmarkus wrote:
Wed Jan 29, 2025 8:57 am
I do have the oil for the 100x objective. I assume a cover glass is used for that, and the oil creates a surface tension droplet between the front element of the objective, and the cover glass?
Even though I have had a 100x from the start, I has been years before I bothered to use it. Well, of course I did try it the first day or so, but without being properly familiar with the microscope and how to handle it, that magnification offers less than satisfactory results. But once you know your microscopy well, then it is very rewarding to make pictures of the smallest what light can see.
iconoclastica,
I think the use of the 100x oil objective will occur in the future. Just wanted to have all the bits required, and more experience under my belt, first.
Jim

jmarkus
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Re: Lighting and DOF ?s

Post by jmarkus »

Been testing focus stacking software (a total of six: Zerene, Helicon, Picolay, ChimpStackr-beta, CombineZP-ran but refused to finish anything, and Lr+Ps). Virtually all of them are faster than Lr+Ps, but I used jpg instead of raw files. However, some do support raw or dng/16bit tif files, and I think that gives a better advantage to color correctness, and shadow/highlight detail. This isn't a proper test, because the 33 stack images are not exposed correctly (2/3rds of a stop under exposed), and I do not think it is fair to draw conclusions from a polished turd. The important criteria to me was #1-Image Quality, #2-Speed, #3-Works over a network (I store my images on a NAS), and #4-Artifacts, because of the time it takes to retouch an image. I am not going to give my ratings on the first three criteria, but one of them dealt with artifacts very differently than all the others. Though Lightroom + Photoshop are not in this comparison photo of artifacts - it looked just like the three that produced so many circular rings or blobs. I'm beginning to think what I thought were artifacts are actually bubbles in the slide cover/slide or medium. I don't know exactly what I am seeing here? This is the same area, but not the main subject. These artifacts appear spread all over the image area, and somehow mostly avoid the center of the field of view.
Attachments
StackerArtifacts.jpg

Pau
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Re: Lighting and DOF ?s

Post by Pau »

Markus, in the posted picture test crops you can't judge at all the stacking software: You're just showing dust stripes produced when the camera and subject move laterally (due to vibrations for example) during the stacking shot.

Better judge the actual subject rendering.

About coupling the Wemacro motor to the fine focus knob, you can take a look at my approach: https://www.photomacrography.net/forum/ ... 22#p232122
Pau

jmarkus
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Re: Lighting and DOF ?s

Post by jmarkus »

Pau,
Thanks for identifying the "artifacts" as dust + vibration. I'm not convinced that is what I am seeing, but I am waiting on a wireless trigger. That should end any potential vibration that may be occurring. The reason I am not convinced is the subject - fresh water plankton - does not have these artifacts. Why would it show only in peripheral areas, and not over the subject? I would think vibration would show across the frame - yet the software has to align and blend the images - so maybe it is extracting data from the central field?

Your magnetic attachment to the fine focus knob is slick. My fine adjustment knob is only 22mm in diameter, is plastic, and about 90 mm off the table. I'll likely build a tray to hold both the WeMicro, and a raised portion for the microscope, stationary, together in one tray. If the WeMicro method is problematic, then I will pursue other methods of attachment.
Jim

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Re: Lighting and DOF ?s

Post by rjlittlefield »

jmarkus wrote:
Fri Jan 31, 2025 10:28 am
I'm beginning to think what I thought were artifacts are actually bubbles in the slide cover/slide or medium. I don't know exactly what I am seeing here? This is the same area, but not the main subject. These artifacts appear spread all over the image area, and somehow mostly avoid the center of the field of view.
As Pau indicated, these are dust trails. Here is some more explanation, copied from https://zerenesystems.com/cms/stacker/d ... hy_is_that ,
My output images have worm trails! Why is that?
That description sounds like what we call “dust trails” or “hot pixel trails”, depending on whether the trails are darker or lighter than their surroundings.

If you carefully check your source images, you will find that in each of them there are a few individual spots, one spot for each trail in your output.

Those spots typically occur at exactly the same pixel coordinates in each source image. (In rare cases individual spots may move slightly as focus is changed, due to axial shift of the lens's exit pupil.)

The reason that trails appear during the stacking process is this: Due to magnification changes, jitter, or systematic drift in your focus stepping setup, the subject moves slightly left/right/up/down in each frame. Essentially the subject is moving around with respect to the spots. In order to make a clean rendering, the focus stacking process must correct for the subject movement, so the subject stays in the same place for each frame. But then the spots appear to move around with respect to the subject. To the focus stacking algorithms, the spots look just like sharp detail that should be retained, and so in the final image you see each spot in every position that it ever occupied with respect to the subject. In combination with the human eye's great ability to pick out linear structures, this causes each spot to become a collection of spots that looks like a straight line, a wiggly trail, or something in between, depending on exactly how the jitter and drift behaved in the first place.
Note that the dots in your images do not change appearance as you stepped focus. That means their cause lies somewhere in the fixed optics, not in the slide or condenser. I'm guessing that they mostly avoid the center of the field of view because some previous owner cleaned that area more carefully.

Pau's point about comparing the subject rendering is critical. The results you're showing suggest that Picolay decided the dust was important and kept that aligned, while letting your intended subject drift around the frame.

--Rik

jmarkus
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Re: Lighting and DOF ?s

Post by jmarkus »

Thanks again Rik. This reminds me of manually stitching photos in Photoshop v3.05 & 4.01 in the mid 1990s using layers and transforms. Picolay must have used transforms to keep all those points aligned, and it did cause the subject to suffer. It had the least desirable image quality of all them. Wonder if Kai Krause (thinking of Kai's Power Goo) could figure out some mathematical way you could stretch and compress the image so it would all be aligned?

You also bring up another question I have about cleaning microscope optics. I would think using Eclipse solution and Pek pads would not harm the coatings? I saw a youtube video on the topic that seemed wrong - in my estimation. Though I do some camera and lens repair - the idea of unscrewing an objective with tiny - possibly cemented doublets, and directional optics makes me nervous. I have been thinking of cleaning my 60x, because it isn't performing well and would provide practice. I assume the internal optics have been in situ since manufacture - so was just going to try cleaning the outer front and rear optics surfaces. If someone has been in there - there is nothing I can do about it.

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Re: Lighting and DOF ?s

Post by rjlittlefield »

jmarkus wrote:
Fri Jan 31, 2025 1:19 pm
Picolay must have used transforms to keep all those points aligned, and it did cause the subject to suffer. It had the least desirable image quality of all them. Wonder if Kai Krause (thinking of Kai's Power Goo) could figure out some mathematical way you could stretch and compress the image so it would all be aligned?
Keeping dust spots aligned is very simple, because they typically never move in the first place. As for keeping the dust spots and the subject both aligned, that would be very weird because those things really do move with respect to each other. Yes, math can be written that keeps them both aligned, but not while also respecting physical reality.
You also bring up another question I have about cleaning microscope optics. I would think using Eclipse solution and Pek pads would not harm the coatings? I saw a youtube video on the topic that seemed wrong - in my estimation. Though I do some camera and lens repair - the idea of unscrewing an objective with tiny - possibly cemented doublets, and directional optics makes me nervous. I have been thinking of cleaning my 60x, because it isn't performing well and would provide practice. I assume the internal optics have been in situ since manufacture - so was just going to try cleaning the outer front and rear optics surfaces. If someone has been in there - there is nothing I can do about it.
Your instincts are good. Eclipse and soft pads should be safe for coatings that can be accessed from the outside. Some other household glass cleaners are distinctly NOT safe, notably anything with ammonia in it. Disassembling objectives is a bad idea. There are a couple of videos on YouTube that show disassembling Mitutoyo 10X and 20X objectives. Those are good for giving some appreciation of what all is inside the barrel, but they're really Really bad for failing to disclose that the objectives never worked properly again.

--Rik

jmarkus
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Re: Lighting and DOF ?s

Post by jmarkus »

Been waiting on equipment to arrive, testing software, and learning how to use both. Still a neophyte, but have learned some of the things most important to me. Speed is proving to be more important than I had anticipated. A proper GUI wasn't something I had considered, but I now find very important. Image quality is still #1, but the two clear software stacking leaders (imo) have different strengths. One is sharper edged and has slightly more noise than the other - which has better chromatic aberration & noise control.

I am really impressed with the WeMicro, and got distracted using it for macro for a few days. Currently I am using my phone to collect the microscope images to stack. Adapting it to the Leica BF200 was simple.

Leica 10x eyepiece, Leica Achro 40x/.65 160/.17, Nikon D7200
WeMicro = Step length 4.0 um, total distance = 1 mm, total steps = 250. I threw away the last 45 images, because they had gone beyond the needed DOF. So, the stack is 205 images. Subject is Spirogyra at 400x. Compared to my first manual attempt a few weeks ago (second image) - I am pleased with the improvement. Still had dust - grrr

Edit - I also lit this more recent attempt both from underneath, and from above using a plain paper diffuser tent & led goose-neck lights.
Attachments
205 image stack
205 image stack
previous 22 image stack
previous 22 image stack

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