40x SPlan woes

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thomas.schwarz
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40x SPlan woes

#1 Post by thomas.schwarz » Thu Mar 26, 2020 12:22 am

Hi friends,

Now on my second 40x SPlan für my Olympus BH-2 BHS.... here is a video I made to demonstrate how poorly my 40X Splan focuses in brightfield and in DIC. Before I posted that it focused poorly in dark field. But it actually focuses very poorly with all illumination and contrast techniques. I have seen sharp videos with 40x SPlan on this model of microscope. So my hope is that there is something that I am missing about the optical planes? Are my slides too thick? I have been using (and in this video I am using CAT NO 7101 Sail Brand slides, 1 mm to 1.2 mm thick, with .13 -.17 mm thick cover glass.... My condenser is a UCD with a "swing out upper lens". I have tried it swinged out and I get horrible focusing on all magnifications.

https://www.youtube.com/watch?v=kVZsqbd ... e=youtu.be

apochronaut
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Re: 40x SPlan woes

#2 Post by apochronaut » Thu Mar 26, 2020 2:20 am

It is always hard to tell what is wrong, when looking at pictures and videos where it seems that there is something wrong. The viewer isn't at the helm, so the standard methods to improve the image aren't available.

Looking at your video, here is what I would do to improve your imaging. The first few recommendations will help overall.

Your imaging is very yellow. This is a common condition with filament type lamps and does reduce the resolution somewhat. Installing a light blue , sometimes called daylight filter over the illuminator window or in a filter holder under the condenser wiĺl improve the resolution with all of the objectives. It is possible under certain conditions that the 40X objective might see a slightly greater improvement than the lower power objectives.

The 40X objective is more sensitive to the position of and the opening of the condenser, than the lower power objectives. This is due to it's higher N.A. The lower power objectives can function normally with a condenser partially closed or backed away from the stage but the 40X, with it's N.A. of .65 or even possibly higher, requires the condenser to be at peak functioning, otherwise there will be a loss of resolution. As a general rule the condenser needs to be close enough to the slide with the 40X in order to establish Koehler illumination. If the microscope does not have Koehler illumination capability, the condenser still needs to be close to the slide: within 2 or 3 mm. The lower the condenser is, the lower the condenser's N.A. is. If it is too low, it will have a lower N.A. than the 40X objective, and reduce it's resolution. This isn't noticeable with the 20X because it probably has an N.A. of .40 or so , and a condenser much lower than the slide will still provide that N.A. to match the objective but with an N.A. of .65 or higher , the condenser position becomes more critical.

Cleanliness. Objective front lens surfaces and rear lens surfaces can be catchment areas for quite difficult to perceive smears that can reduce resolution and or contrast. The problem can be really hard to see . Being careful to immaculately clean those two critical lens surfaces is important.

With second hand microscopes.
The 40X has only a slightly greater working distance than an oil immersion lens. It is not uncommon for a 40X to have been frequently dipped into immersion oil, where an immersed objective is also frequently used. It happens by accident during objective rotation but the 40X has no oil seal. The oil can creep into the lens and the first symptom of oil invasion is a loss of resolution. Some 40X objectives are more susceptible to oil invasion than others.

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Re: 40x SPlan woes

#3 Post by PeteM » Thu Mar 26, 2020 2:23 am

Three things to try:

1) Have you thoroughly cleaned the front element of the 40x SPlan and inspected it under magnification? It will take a small cotton swab and a proper lens cleaner. Could be the objective has a lifetime of junk on it; commonly some immersion oil.

2) Did you adjust the Kohler illumination from 20x to 40x? Any off-center or stray light can have an effect.

3) Have you tried a very thin sample for comparison, rather than sand etc..? There's much less depth of field at 40x.

On edit - Apo has covered much of this while I was reading & typing.

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Re: 40x SPlan woes

#4 Post by BramHuntingNematodes » Thu Mar 26, 2020 3:25 am

I had a high dry objective that I couldn't quite get sharp focus from and found the coverslip thickness was the problem. I believe the Olympus standard is .17, and so you would need to find slips in the highest thickness of your stated range to be compliant, while the mean assuming a uniform or otherwise symmetrical distribution of slip thickness is well below compliance. It might not help, but is among the cheapest and simplest solutions.
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Re: 40x SPlan woes

#5 Post by deBult » Thu Mar 26, 2020 4:06 am

My 40* SPlan (non Apo) is very sensitive to correct coverglass thickness. Zeiss sells 0.17 +- 0.005 coverglasses: expensive but they do make a difference.

Best, deBult

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Re: 40x SPlan woes

#6 Post by ImperatorRex » Thu Mar 26, 2020 5:12 pm

Dirt or sand is not really a good sample to test. The layer beyond the cover glass is most likely already too thick for getting good quality image. For DIC why not just use the epithel cells from your mouth? Just grab some stuff with your fingers from the mouth insides and smear it on the objects carrier, a bit water and the coverglass. That is all. Should give you nice, thin test sample.

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Re: 40x SPlan woes

#7 Post by thomas.schwarz » Fri Mar 27, 2020 7:38 pm

great feedback and tips. thank you so much. i tried some of the ideas including extensive cleaning of objectives and condenser. of course there is always more that one can do, but with just the objective and 80% of the condenser lenses, there is some small improvement! thank you.

then about the distance of the condenser to the specimen, what i did was to switch out my UCD for the original condenser that comes with my BH2 BHS. there, i notices what i think is called the kohller Illumination, meaning the other diaphragm directly on the light source. there was again a small, but noticeable improvement by getting just the right amount of diaphragm closure there, combined with the right amount on the condenser.

I am using the coverslides with .17 to .13 thickness. so in short, there is still a lot more for me to experiment with. but... ..for motivation, could someone please post a video of BH2 40x that is well focused?

Thanks!

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Re: 40x SPlan woes

#8 Post by 75RR » Fri Mar 27, 2020 11:08 pm

I am using the coverslides with .17 to .13 thickness.
Depending on the source, the thickness stated on the cover slip box may vary quite a lot.

40x objectives and above perform better when supplied with the cover slip thickness that is printed on them.

If it says 0.17mm - I try to give it 0.17mm

I use an old Moore & Wright 961MB with an accuracy of 0.002mm. They can be picked up on Ebay with a little patience for reasonable money.


For best results the subject you are imaging should be placed up against the underside of the cover slip.

This requires that the cover slip and the slide be very close to each other.

Avoid putting too much detritus on the slide, if you can see it with the naked eye you have probably overdone it.


Note: I reuse cover slips, in fact I tend to use them until they break or the scratches drive me crazy.

The point of this story is that I don't have to measure cover slips every time I use my microscope.

I just do it when I run out of 0.17 and 0.16+ cover slips.

Then I measure enough of them in order to be able to set a few aside - say once every couple of months or so.

https://www.ebay.co.uk/sch/i.html?_from ... 1&_fosrp=1
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Re: 40x SPlan woes

#9 Post by thomas.schwarz » Sat Mar 28, 2020 12:28 pm

super interesting discussion.... there is a lot that i can do to prove out the theory/hypothesis, that my current focusing woes are significantly impacted by cover slide thickness.... how about using a prepared slide? just did that... ...so there i have no idea about slide thickness (doah!) but surely there are no oceans of water sloshing around, as often happens with my greedy desire to see more and more microbes in one slide!!!!! Action please!!! ....and yes, it focuses fine with brightfield as well as DIC 40x S Plan. So I will focus on coverglass thickness and smaller single-water-drop samples and see how good of a focus I can get....

....short question dazu, what about a modern micrometer? Do you, like me, just like the quality/ look and feel of older things? Or do you think that this/or similar modern ones will not measure well?
https://www.pollin.at/p/digital-mikrome ... plEALw_wcB

that one should measure to an accuracy of 1/1000 of a mm which should be enough, yes?

Another whole take on this subject is resolution. It still seems like I am just zooming in and not increasing resoltuion/ not seeing new details. So any videos showing increasing resolution with well focused objective of increase magnification would be very helpful.... 4x to 10x to 20x, clear I can see more details with each magnification increase. But 20x to 40x, even with good focus, it feels like I am looking at the same digital image with zoom, e.g. no increase of resolution....

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Re: 40x SPlan woes

#10 Post by 75RR » Sat Mar 28, 2020 1:21 pm

Not necessarily advocating buying from Amazon, but I find the comments left by buyers useful https://www.amazon.de/B%C3%BCgelmesssch ... NrPXRydWU=
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Re: 40x SPlan woes

#11 Post by Roldorf » Sat Mar 28, 2020 1:47 pm

Just when you need it, the battery will be flat.
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Re: 40x SPlan woes

#12 Post by PeteM » Sat Mar 28, 2020 8:31 pm

FWIW, it used to be that the batteries in Mitutoyo digital micrometers and calipers would last one or two years, while those in Chinese knock-offs might be gone in a month. Mitutoyo took great care to have low drain circuits, automatic shut-off etc. Just a look at the circuit boards also showed a tremendous difference in quality.

The battery-drain situation has improved in recent years. - likely with the micrometer listed. Personally, I'd consider getting a Mitutoyo digital caliper. It would be accurate enough to measure cover slips and would come in handy for all sorts of other uses (parfocal distances, thread sizes, difference between 30 and 30.5mm stereo eyepieces, etc. etc.).

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Re: 40x SPlan woes

#13 Post by 75RR » Sat Mar 28, 2020 9:22 pm

I have both a micrometer and a caliper but it is true that a caliper can do both jobs at a pinch,

particularly if it is possible to see the difference between two measurements even if it lacks a vernier.
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Re: 40x SPlan woes

#14 Post by Hobbyst46 » Sun Mar 29, 2020 10:14 am

75RR wrote:
Sat Mar 28, 2020 9:22 pm
I have both a micrometer and a caliper but it is true that a caliper can do both jobs at a pinch,

particularly if it is possible to see the difference between two measurements even if it lacks a vernier.
I think that for measuring thin items, a micrometer is more reliable simply because the contact area is larger.
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Re: 40x SPlan woes

#15 Post by thomas.schwarz » Tue Apr 07, 2020 9:13 pm

Hi just wanted to follow up here... ...using a very thin specimen, just one little drop of water, I do have improved resolution and focusing with my 40x objective. please let me know if you think that this is about as good as it gets? or if I should get more resolution? I am still waiting for my calibration slide, so that will come later....

https://www.youtube.com/watch?v=aL6YJNQ ... e=youtu.be

my best guess, is that I can continue to clean various lenses, measure cover glass thickness and so on to get some additional resolution at 40x.... thanks again for the help and feedback.

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Re: 40x SPlan woes

#16 Post by deBult » Wed Apr 08, 2020 1:54 am

Well, no.

I’m always amazed at the perceived drop in image quality when switching from 20* to 40*, but it looks like your suffering from colour fringes around your subject.

Try to get the Zeiss coverglasses mentioned earlier (with the tight tolerance thickness).

Best, deBult (relative beginner with a BH2 and SPlan optics).

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Re: 40x SPlan woes

#17 Post by 75RR » Wed Apr 08, 2020 6:09 am

thomas.schwarz wrote:
Tue Apr 07, 2020 9:13 pm
Hi just wanted to follow up here... ...using a very thin specimen, just one little drop of water, I do have improved resolution and focusing with my 40x objective. please let me know if you think that this is about as good as it gets? or if I should get more resolution? I am still waiting for my calibration slide, so that will come later....

https://www.youtube.com/watch?v=aL6YJNQ ... e=youtu.be

my best guess, is that I can continue to clean various lenses, measure cover glass thickness and so on to get some additional resolution at 40x.... thanks again for the help and feedback.
Ideally you would use a diatom test slide to check resolution. It is rather hard to say how much better your resolution can be, based on a video - too many variables.

http://www.microscopy-uk.org.uk/mag/ind ... -test.html

Something like this 8 Form Test slide by Kemp is very useful:
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Re: 40x SPlan woes

#18 Post by ImperatorRex » Wed Apr 08, 2020 3:29 pm

Agree with 75RR, the test diatom plate would be very helpfull. You can use for example Pleurosigma angulatum very good to check resolution of a 40x/0.65 n.A. Objective. With DIC and the full opened aperature you should not have issues to resolve the structure of the frustel.

But quick sample could also be to taken for epitel cells inside your mouth - see my previous thread.

It should give you somethin like below:
https://www.mikroskopie-forum.de/index. ... #msg115967

Foto below was even taken with an very old and poor camera:
https://www.mikroskopie-forum.de/index. ... #msg115978

Edit: some guidance how to use such a tesplate is described here:
viewtopic.php?f=18&t=6905&p=61417&hilit ... tum#p61417
(can provide the original german text if someone is interested)

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Re: 40x SPlan woes

#19 Post by apochronaut » Wed Apr 08, 2020 10:40 pm

I have been following this along and am in general agreement that probably the objective is suffering from spherical aberration but there is still something nagging me , though. This is a plan achromat 40X .70 objective, is it not?
It's kind of unusual for an objective of such a modest N.A. to need such fussing over the cover slip. I've used probably 100 to 150 such objectives over the years and with objectives of between .55 and .75 I have never seen that before. Maybe when you get above .75 without a collar, the tolerances get tight with some but I have a 40X .80 planapo without a collar that can focus well with a .19 cover slip. Same with a 40X .70 planfluor. It lets you know it's having focusing problems when the objective hits the cover slip. If it's got clearance at all the focus is acceptable.

Is it possible Thomas, that you have been floating the cover slip? Even then , if it is less than .20 , you should get good focus in the upper zone of the sample.
You earlier said your cover slips were loosely graded at between .13 and .17. I can't see how you would run into the problems you have been having with covers in that range and a relatively pedestrian 40X objective.

The fact that you found that you are getting better focus with a prepared slide is also odd because typically, prepared slides unless they are very expensive ones , use average grade cover slips and any used in schools where the microscopes have a .65 N.A. high dry objective have thicker than normal cover slips in order that they offer a bit of resistance in the event of an objective contact. I've seen .22 or even greater covers on lots of prepared slides and an average 40X objective should not have the kinds of problems you are having. That's one of the reasons average microscopes have a 40X of .65 to .70 N.A., so sample thickness problems aren't an issue.

It doesn't seem like cover slip thickness is your only problem.

Can you post a picture of what you consider to be good focus with a prepared slide?

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Re: 40x SPlan woes

#20 Post by BramHuntingNematodes » Thu Apr 09, 2020 12:47 am

Hmm yes the objective I had problems with was a .85 fluorite, and anyway I didn't then have to bust out the micrometer. Rather I ditched my cheapo amscope slips that were marked .13 and bought some others marked .17 (note this was a B&l so .17 is still .01 mm too thin).
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Re: 40x SPlan woes

#21 Post by MichaelG. » Thu Apr 09, 2020 6:59 am

thomas.schwarz wrote:
Tue Apr 07, 2020 9:13 pm
Hi just wanted to follow up here... ...using a very thin specimen, just one little drop of water, I do have improved resolution and focusing with my 40x objective. please let me know if you think that this is about as good as it gets? or if I should get more resolution? […]
I don’t feel qualified to advise, Thomas ; but will just comment:
It appears to me that the 40x part of your video illustrates a significant problem with chromatic aberration.

The fringing looks excessive, and seems to vary a lot with focus.
... I suspect a mis-match of optics. [*]

MichaelG.

.

[*] the lenses themselves and/or their relative locations
___ I seem to recall you having some difficulty with your camera fittings.
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Re: 40x SPlan woes

#22 Post by MichaelG. » Thu Apr 09, 2020 8:46 am

I searched for images which might set a reasonable expectation for you, Thomas
... and found this: http://www.microbehunter.com/forum/phot ... a-zygnema/

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Re: 40x SPlan woes

#23 Post by apochronaut » Thu Apr 09, 2020 10:36 am

I agree. There is an unacceptable level of ca as well as a foggy quality, which doesn't really conform to a spherical aberration problem. What relay lens are you using for the camera? On another note; is the problem the same with the eyepieces, or is it just the video.

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Re: 40x SPlan woes

#24 Post by ImperatorRex » Thu Apr 09, 2020 12:27 pm

Thomas, it looks like the video was done with DIC and some higher order of difraction, so the DIC has this color contrast.
So the color halos or gradients observed may be the result out of that?
I would propose to test with conventional bright field first - what may help to exclude that the issue is not related to some DIC incompatibility?

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Re: 40x SPlan woes

#25 Post by thomas.schwarz » Mon Apr 13, 2020 6:57 am

hello friends,

first off thank you very much for your help. I think that this is a very complex domain (validate correct and optimal performance/resolution for a given microscope).

in short, i will order the diatom slide and follow up, starting with bright field. but in parallel, lets see if my optics/lenses are basically correct.

I did clean my projection eyepeace, Olympus NFK 2.5X LD. 125, but I also noted that I think I have the same problem in binocular view, as well as phototube viewing. I say "think" because they are hard to compare, because my binocular view is a much, much larger field of view, and phtototube (with panasonic gh4) is a much smaller field of view, and much bigger image.

* my 20x and 40x objectives are SPlan 0.70, 160/0.17. Is that correct for bright field and for the standard bh2 bhs?
* my condensor is the universal condenser. but i have the same problem focusing my 40x with my standard/stock condenser.
* my phototube is the standard original olympus phototube

I did make a slide with mouth cells, and clearly(?) i have the same problem there.
https://www.youtube.com/watch?v=CLlEBMfIYfE
https://www.youtube.com/watch?v=KmYqT5obcUU

and regarding the algae reference image from micheal g, yes, my 40x has much worse resolution.

thanks!

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Re: 40x SPlan woes

#26 Post by apochronaut » Mon Apr 13, 2020 11:16 am

Your objective specification for the 40X , seems to be correct. Has a rubber grip ring?

Just a couple of things to check and or rule out. 1) Given the stated difficulty in comparing the visual image with the camera image, try putting one of the eyepieces in the photo tube( if it is possible..I know that they are quite broad in physical profile and I don't know the structure of a BH-2 photo tube well) and compare the two images with the same eyepiece. That should rule out something adverse in the trinocular that could disproportionately affect the 40X. Not likely but possible.

The Alan Wood site mentions that the correct distance between the eyepiece rest and the sensor is 150mm, for NFK eyepieces.

I know since this is the second 40X objective offering up the problem, that it is unlikely to be the objective itself but I would still have a look inside the objective very carefully. Sometimes an objective design is prone to certain problems, so the same problem has a tendency to crop up in various examples of it. If you look through the back of the objective , while aimed at a broad illumination source like a window, rotate it slowly, all the way around several times. Is there anything moving in there? Small amounts of invaded immersion oil, will cause the kind of problem you are having and it's always possible that two objectives in a row could be fouled.
If you can get access to a stereo microscope that has a transmitted illumination feature, you can focus down through the lens pack from back to front and then front to back and see if there is any de-lamination.

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Re: 40x SPlan woes

#27 Post by thomas.schwarz » Sat Apr 18, 2020 11:27 am

Thanks for the feedback. So I am simplifying the problem domain by sticking to only bright field. And also in this video, showing the specimen with all obectives: 4x dplan, 10x dplan, 20x splan, 40x splan. My next step will be to try the regular eyepiece in the photo tube, look more into the objectives for dirt/oil intrusion, and then to open up the trinocular head completely tonight and clean it with rosignol lighter fluid and quetips.

One other totally unscientific observation, but I have to say it because it is so true.... ...call it subjective (?), but some days its seems so much better and some days so much worse. I mean, take a look at these videos from today. Then take a look at another from a month ago.

From today (and for the purpose of this thread):
pt 1 https://www.youtube.com/watch?v=7YM2JoQNzd4
pt2 https://www.youtube.com/watch?v=SIkRlc4ScBo

From a month ago (for comparison):
https://www.youtube.com/watch?v=C2bPJvfzxSU

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Re: 40x SPlan woes

#28 Post by thomas.schwarz » Sat Apr 18, 2020 10:08 pm

major major break through here. issue fixed by shortening photo tube by 30% (was 15 CM now about 10). I had the OEM photo tube that came with the microscope and was used for that 35 MM Film Camera. Fix was to go (back) to the MTV-3 Olympus photo tube. And interesting, that photo tube came with a lens (works in addition to the photo projection eyepiece/higher in the light path), and I achieved optimal resolution with that lens removed, so just the photo projection eyepiece. Any optics experts out there know what the lens in the MTV-3 was for? Was it assumed that the MT>V-3 would work without the photo projection eyepiece? ...would love to know how the microscope performs with that MTV-3 lens, without the photo projection eyepiece... but I will never know because I superglued my adapter on to the MTV-3....

Here is a video of the result.
With additional lens in MTV-3
https://www.youtube.com/watch?v=51zRtBTFrSs

Without additional lens in MTV-3
https://www.youtube.com/watch?v=EJEdlvs28pI

Look at same tiny algae at 100x (oil with cover glass) with DIC
https://www.youtube.com/watch?v=wuqUQkj0fq0

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Re: 40x SPlan woes

#29 Post by Scarodactyl » Sat Apr 18, 2020 10:32 pm

The mtv lens is a reducing optic. It is an essential part of that adapter for using small sensor cameras. If you remove it you've turned a valuable and useful adapter into a simple dovetail adapter.
If I were you I'd bust out the acetone, remove the superglue, reassemble the mtv. Sell that adapter to one of the many poor olympians out there who want to use a small format camera (the cheapest complete one on eBay is like 150usd, you could probably get 100 quickly). In the mean time take a simpler, cheaper approach to parfocal adaptation of your camera, (estimated cost around 35 bucks) then buy something nice with the profits.
Edit:here's a diagnostic instruments adapter for yout scope that just came up for sale mislabeled, so you can have form and function cheaply if you want https://www.ebay.com/itm/233563794595

I am still confused though. Setting up a dslr on a bh2 is very easy if you have the projective--the camera just has to sit the right distance above it. Did the other adapter have additional optics in it or something? If you had it at the wrong height with no intervening optics I think the objectives shouldn't have been parfocal anymore.
Last edited by Scarodactyl on Sun Apr 19, 2020 8:16 am, edited 1 time in total.

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Re: 40x SPlan woes

#30 Post by deBult » Sun Apr 19, 2020 7:25 am

Still wondering about the impact of coverglass thickness variation though: different coverglasses used?

As stated my SPlan 40 is sensitive to coverglass thickness variation.
In my old Olympus G-type mono you could adjust the tube length to compensate for coverglass variations: but this was only mm.

Best, deBult

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