Hole in my microscope...

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neal Shields
Posts: 58
Joined: Sat Jun 04, 2022 8:02 pm

Hole in my microscope...

#1 Post by neal Shields » Sun Nov 27, 2022 4:59 pm

I purchased a microscope from a retired research scientist.

After owning it a while I realized that once he decided what he wanted to do with a microscope and which brand, he had very few, if any, options for an optimal (for him) configuration. As when he purchased my microscope he had been working as a scientist for about 30 years, he knew how to buy a microscope.

His primary consideration was obviously ease of use. He needed both DIC and Phase and the ability to switch back and forth quickly.

That drove his objective decision. He used semi-apochromatic FL objectives that had phase rings.

The invoice says of these objectives: "for brightfield, dark field, phase contrast, Nomarski DIC, polarization and fluorescence". That kind of covers it.

Although he didn't seem budget constrained there were no APO objectives that covered all these bases. (No phase available)

However, although the manufacturer says that you can use these objectives for DIC they acknowledge that the unused phase ring limits DIC results. Under "DIC" they state "limitation" where the APO lens says: "excellent".

Having said that I am getting great (in my opinion) results using these objectives for DIC. My microscope photography is improving but I still only get about 80% of what I can see to the "film".

These are three photographs taken at 400x of blood cells in tissue at three different DIC prism settings.

https://www.flickr.com/photos/196001110 ... ed-public/

https://www.flickr.com/photos/196001110 ... ed-public/

https://www.flickr.com/photos/196001110 ... ed-public/

When I purchased the microscope it had three objectives UplanFL phase 10x, 40x, and 100X.

So now my problem: I had two empty holes in the objective turret.

I have filled one with an old 4x 160 although my microscope is infinity corrected. At that magnification it doesn't seem to matter especially as I only use 4x to orient myself on the slide before going to 10x or above.

So what to fill the last hole with?

I thought about a 60x because I hate cleaning up oil. Not much of a problem unless you are OCD (I am) so it must be CLEAN, not just wiped down.

However I have some 15x eyepieces and looked at a chart that said that I had more than enough resolution with my 40x to use a 15x eyepiece with it. That kind of made a 60x redundant.

I considered a 100x APO but I hate oil and more importantly I got on the Nikon site and found a chart that shows that going from an FL to an APO at 100x oil only increases resolution from .21 to .20 nanometers. Unless you are looking for early signs of cancer for a living, I can't see that that justifies the exorbitant expense if you already have the FL.

Now I am looking at a 40x apo that takes my 40x resolution from .37 to .29 (kind of).

My dry condenser lens only goes to .9 and the APO objective has an NA of .95 so I would only get about 3/4 of the additional resolution (The FL lens has an NA of .75).

Can anyone comment BEFORE i spend the money?

I am also confused as to why such a small difference in the 100X range. This is the link to the Nikon site.

https://www.microscopyu.com/microscopy- ... resolution

One other question that may be too esoteric for anyone to answer. My current 40x Nomarski prism is an Olympus U-DP40. The spec sheet for the APO lens calls for a U-DPA40. I assume the "A" stands for APO and just means that it is manufacturered to a higher tolerance? (Or maybe it is built the same and is selected? That was the way we got "high temperature" transistors back in the day.)

I assume my U-DP40 will work but if anyone knows better please let me know.

Neal

apochronaut
Posts: 6269
Joined: Fri May 15, 2015 12:15 am

Re: Hole in my microscope...

#2 Post by apochronaut » Sun Nov 27, 2022 6:33 pm

Resolution isn't the only specification to consider in choosing objectives. Calculating theoretical resolution is just what it says : theoretical. Colour correction and contrast anomalies can mask or obscure resolution , so the apochromats of any given microscope objective family will provide better realization of theoretical resolution than less well corrected members of the family.

A .90 condenser will allow for an N.A. above 1.00 if the objective is working above 1.0.because the final N.A. is a product of the below object and above object optics.Typically, a 1.25 N.A. oil immersed objective for instance will work at around 1.12 with a .90 condenser. A 1.35 N.A. objective , higher.

Definitely seek out a 20X plan fluorite objective, or achro or apo. You should be able to find an Olympus branded one at an acceptable price but the Olympus formatted PlanF from China or India are pretty good and cheap. Q.C. issues can be a factor sometimes with modern new stencil brand objectices. Poorly performing ones will get replaced but having to do so is annoying. In my experiemce in a lot of 10, about 2 aren't up to spec. That fraction is lower with higher spec. objectives, which get closer attention.

I have some fully tested Olympus formatted 100X 1.15 water immersion 100X objectives I am selling. Plan Achromats. While these are in no way going to perform up to the standards achieved by your uplanFl, the water immersion can sometimes be useful.

neal Shields
Posts: 58
Joined: Sat Jun 04, 2022 8:02 pm

Re: Hole in my microscope...

#3 Post by neal Shields » Sun Nov 27, 2022 9:08 pm

I am really tempted by a water immersion objective. I assume those can be used to study pond water?

I have gotten side tracked by a great set of histology slides I got cheap, but expect to get back to pond water soon.

Thanks for the information on objective NA vs Condenser. Since i posted I found some "official" information that confirms exactly what you say.

It surprised me that the condenser didn't match the objective because in every other case the microscope was configured perfectly and I feel I really owe a debt to its first owner.

Thanks for the quick reply

Scarodactyl
Posts: 2775
Joined: Sat Mar 03, 2018 9:09 pm

Re: Hole in my microscope...

#4 Post by Scarodactyl » Sun Nov 27, 2022 11:36 pm

I'd aim for a 20x. The uplanfl 20x is quite good and the 20x uplansapo is even better in image quality, and sometimes (though uncommonly) comes up at low prices.
The water objectives are also very enticing, I still need to get one of those.

PeteM
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Location: N. California

Re: Hole in my microscope...

#5 Post by PeteM » Mon Nov 28, 2022 12:13 am

FWIW, the UPlanFl phase objectives aren't bad for DIC, but I believe you'd notice an improvement without the phase rings.

If you're interested in a 20x UPlanSApo, I have a spare (lucked into a two-for-one-price at just above the usual price for one). Both provide good images and I could likely sell one for around 2/3rd the typical Ebay prices.

apochronaut
Posts: 6269
Joined: Fri May 15, 2015 12:15 am

Re: Hole in my microscope...

#6 Post by apochronaut » Mon Nov 28, 2022 2:31 am

neal Shields wrote:
Sun Nov 27, 2022 9:08 pm
I am really tempted by a water immersion objective. I assume those can be used to study pond water?

I have gotten side tracked by a great set of histology slides I got cheap, but expect to get back to pond water soon.

Thanks for the information on objective NA vs Condenser. Since i posted I found some "official" information that confirms exactly what you say.

It surprised me that the condenser didn't match the objective because in every other case the microscope was configured perfectly and I feel I really owe a debt to its first owner.

Thanks for the quick reply
Water immersion objectives are of two types. Cover slip corrected and dipping. Dipping objectives have had a flurry of interest during several epochs since microscopy designs and specifications reached some degree of standardization around the turn of the 20th century. The object medium needs to be pretty still. Currently they are favoured for some cellular biology applications.
They don't work very well for pond water but cover slip corrected objectives do. Although oil immersion objectives are capable of achieving very high N.A.s, the mismatch in refractive index between water and immersion oil gets problematic in depth the farther one descends below the cover slip, so while most corrections are maintained, spherical aberration raises it's fuzzy head. Less so with water objectives which achieve a fuller performance matching their N.A. into the sample depths.
What is missing are inexpensive planfluor or planapo water immersion objectives. In the used market, only glycerin immersion shows up as a low cost option in any availability.

neal Shields
Posts: 58
Joined: Sat Jun 04, 2022 8:02 pm

Re: Hole in my microscope...

#7 Post by neal Shields » Mon Nov 28, 2022 10:18 pm

Water objectives...

This may be the last objective I ever buy (unless I find a steal at an estate sale or thrift store) so I want to get it right.

Further I really don't have a problem that needs fixing. If I hadn't had a new lens in my right eye about 3 years ago all of this would be a moot point but with NO cataracts I can see differences that I haven't been able to see for 30 years.

At this moment I am down to either:

Uplan S APO 40x dry NA.95 (the .85 makes no sense as it is too close to what I already have)
or
UAPO/340 water NA 1.15

With my .9 condenser the dry gives me a resolution increase of 10.7% over my 40X Fl
The water gives me a resolution increase of 19%

So in the old, in for a penny, in for a pound logic...

Three big questions I have are:

I assume the "340" means it is optimized for fluorescence?
Does that negatively impact ordinary light microscopy? The data sheet says brightfieqld is "excellent" and DIC is "excellent".
Most of all if I use it dry will it be better or worse than the 40X NA .95 dry APO? If not there is no logic to not getting the water.

I am down to these two choices because i don't want to complicate the use of my scope. I have a 5 position turret with all the holes filled: Ph2,Ph3, DIC 10,40,100.
(For normal brightfield the manual says to use the 100x DIC position. That worried me for several months until I read the instructions.)

If I am going to spend this much money I want the DIC capability.

My goal is to get as much magnification as I can get without using oil. With even my existing 40x I can used a 15x eyepiece but not 20. With either of these I should be able to get away with a 20x eyepiece so would be up to 800x w/o oil.

All of the numbers assume that the phase rings are not costing me anything when I use DIC and I know that isn't the case (from the data sheets) so my DIC gain at 40x may be more than just comparing a .75 NA to a .95 or 1.15 NA objective.

Comments and advice appreciated.

I have a lot of time to make a decision as I am selling off some surplus parts on eBay and those sells are intended to fund this purchase.

Thanks
Neal

apochronaut
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Joined: Fri May 15, 2015 12:15 am

Re: Hole in my microscope...

#8 Post by apochronaut » Tue Nov 29, 2022 7:31 pm

20X eyepieces are impractical. They used to be an option for use with apochromats sporting very high N.A.s but that was when the eye relief of most eyepieces had limitations and the extra limitation that a 20X imposed was not that much more. Modern 10 and 15X tend to be very good for eye relief but 20's not consistent with them, usually. Not that they couldn't be but there isn't much of a market and a high eye relief 20X with a wide flat field would be a big ticket item.
Their main value is in using high N.A. low magnification objectives at very high total magnifications and thus preserving a relatively long working distance.
Using a 10 at 200X instead of a 20X would normally, greatly increase the working distance.

Scarodactyl
Posts: 2775
Joined: Sat Mar 03, 2018 9:09 pm

Re: Hole in my microscope...

#9 Post by Scarodactyl » Tue Nov 29, 2022 7:41 pm

Yeah, they still get some use on stereo microscopes--I got a pair of 25xes on a leica head recently. They have a wide apparent field and give as good an image ad can be exoected but you'd need a really specialized task for then to be worthwhile.

Dubious
Posts: 426
Joined: Sun May 09, 2021 7:55 pm

Re: Hole in my microscope...

#10 Post by Dubious » Tue Nov 29, 2022 9:09 pm

If it were me, I'd aim for a 20x objective--a very useful power for water critters.

10x eyepieces are the standard for most systems. Resolution is largely determined by the numerical aperture of the objective, and increasing the magnification of the eyepiece is likely to result in little more than empty magnification without increase in resolution.

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