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.

PeteM
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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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deBult
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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.

thomas.schwarz
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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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75RR
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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.

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thomas.schwarz
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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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75RR
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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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Roldorf
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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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PeteM
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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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75RR
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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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