Is Dark Field Possible at 100x with Immersion Oil?

[Sure Squintsalot]

So that I don't tear out what's left of my hair....

Is trying to get darkfield at 100x with immersion oil a fool's errand?

I can't blame my set-up, it's pretty reasonable (I think):

• Nikon 100x/160 0.8-1.30 N.A. UV-F Glycerin Immersion objective

• Nikon flip-out Achromat Condenser 0.9 N.A.

• Standard slide thickness, 0.17mm coverglass, plankton permanent mounts

You've done it? Looks great? Let's see!

[PeteM]

I'd blame your setup.

It sounds like you're trying to use something like a darkfield stop in a .90 na condenser and hoping that if you stop the objective down to .80 na, you'll be OK? With great care, great luck, perfect centering, etc., you might get a sort of darkish background.

You'll want a proper darkfield oil immersion condenser (numerical aperture more like 1.30) with the slide oiled top and bottom and (likely) the objective still stopped down quite a bit.

The UV-F glycerine objective may also not be ideal. It was designed primarily for UV fluorescence work. It could be that it's not ideal (loss of contrast) for darkfield.

[Sure Squintsalot]

I'd blame your setup.

It sounds like you're trying to use something like a darkfield stop in a .90 na condenser and hoping that if you stop the objective down to .80 na, you'll be OK? With great care, great luck, perfect centering, etc., you might get a sort of darkish background.

That sums it up....but my background gets barely dimmed, even with the condenser touching the slide. I don't think I've ever seen a Nikon oil immersion condenser, at least not for finite systems.

[PeteM]

They're out there. Sometimes a previous generation Nikon "Ultra Dark Field" condenser shows up fairly cheap. This won't have a dovetail attachment at the bottom - but that can be added (either machined and screw-attached or 3D printed and epoxy attached).

[apochronaut]

Your problem is the differential between the objective and condenser. Your objective stops down to .80 and your condenser is .90. Theoretically the objective's N.A. is lower than the condenser's which is necessary but it isn't lower by enough. Flare and dispersion cause the DF to be greyfield. Using a dedicated oil immersion DF condenser which will have a minimum N.A. of greater than 1.10 will provide DF and at a higher N.A.
You can only do so much with stops in a refracting confenser. Seldom is DF above 40X .65 possible

[Hobbyst46]

This post from the past
http://www.microbehunter.com/microscopy ... SER#p70759
Demonstrates that by pure chance, a 100X1.25 objective can yield an (imperfect) dark field. A dedicated 1.2-1.4 NA immersion DF condenser was used. Of the three 100X iris-less objectives that I tested, DF was achieved from only one. Apparently, the tiny hole that exposes the front lens of that objective functions as an effective iris in this application. Just an additional small fraction of NA made the difference. Naturally, it would allow relatively little light inside, so bad for fluorescence...

[Phill Brown]

It can't work with a low power condenser.
0.8 na is possible but as said it's challenging,a phase telescope would be very useful to cut out the guess work and lost hours,maybe pick up a 1.25 condenser and modify it to DF.
Nikon use an acrylic lens in the labophot era condensers.
Edit: Had a go with Nikon 1.25 abbe.with the nearest objective I have which is 50x .95 oil Fl.
Couldn't get an image with phase telescope on the plane a patch would need to go to check what size would work.
Using a flat glass with rings marked on with a fine tip marker.
It's not a Nikon objective which might not help.
I'll leave the experiment there with the conclusion that if it can be made to work the results will be disappointing compared with x40 .65.
Possibly consider x60 .8.
Good luck anyway.

[Sure Squintsalot]

Thank you all for your learned replies.

I'll set aside dark field ambitions for this objective, for now.

It does, however, seems to work reasonably well with my DIC set-up.

For anyone else reading this, spend the extra money on some decent glycerin immersion oil. The food-grade stuff sold by Whole Foods is absolute kah-kah.

[apochronaut]

Immersion oil needs to be 1.52 refractive index. You can use corn oil or something like that but there will be spherical aberration.

[PeteM]

FWIW, in Sure Squints.. case, his fluorescence 100x objective is designed for the lower R.I. of glycerine immersion. It doesn't achieve the highest numerical apertures of oil immersion, but the glycerine is more or less transparent to UV.

[apochronaut]

Sorry. I knew that Mind went into neutral. That 100X objective Nikon objective actually works well in a Diastar.

[apochronaut]

This post from the past
http://www.microbehunter.com/microscopy ... SER#p70759
Demonstrates that by pure chance, a 100X1.25 objective can yield an (imperfect) dark field. A dedicated 1.2-1.4 NA immersion DF condenser was used. Of the three 100X iris-less objectives that I tested, DF was achieved from only one. Apparently, the tiny hole that exposes the front lens of that objective functions as an effective iris in this application. Just an additional small fraction of NA made the difference. Naturally, it would allow relatively little light inside, so bad for fluorescence...

Is it possible the objective had an unoriginal rear diaphragm? One with a slightly smaller hole, or is the rear aperture on that a fixed aperture...some don't thread out.

[Phill Brown]

With a zonal oil DF condenser with an oil objective it's still a requirement to use stop down to 0.9 or less to get a dark background.
Unless I see it I'll take that as the limit.
It's possible to get fair results dry at 0.8 but not with a low power condenser.

[woyjwjl]

I cannot guarantee whether the following 'English' is logical:

High numerical aperture reflective condenser and dark field illumination provide the preferred method for observing and capturing very small collections of particles or colloidal suspensions, even if the particle diameter is significantly lower than the resolution limit of the objective lens. This is due to the diffraction light of particles, which becomes visible through the objective lens as a bright diffraction disk. As long as the lateral distance between adjacent particles is greater than the resolution limit of the objective lens, each particle can be seen as a tiny diffraction disk. As the illumination intensity increases, the optical difference between small diffractive particles and their background also increases. At the same time, even smaller particles (detected solely by their ability to scatter light) can now diffract enough light to become visible, even when the diameter of suspended particles is less than 40 nanometers, which is approximately one-fifth of the 200 nanometer resolution limit of the highest numerical aperture oil-immersed objective. In biological applications, using a high numerical aperture dark field condenser can observe and capture the movement of live bacterial flagella with an average diameter of about 20 nanometers (too small to be seen under bright field or DIC illumination).

Personally, I think that the dark field condenser is very professional (minority), and amateur should not set foot in it.

Anyway, this is in my favorites, priced at $400 (excluding shipping). Do you know if OP is interested?

Best Regards

[Hobbyst46]

This post from the past
http://www.microbehunter.com/microscopy ... SER#p70759
Demonstrates that by pure chance, a 100X1.25 objective can yield an (imperfect) dark field. A dedicated 1.2-1.4 NA immersion DF condenser was used. Of the three 100X iris-less objectives that I tested, DF was achieved from only one. Apparently, the tiny hole that exposes the front lens of that objective functions as an effective iris in this application. Just an additional small fraction of NA made the difference. Naturally, it would allow relatively little light inside, so bad for fluorescence...

Is it possible the objective had an unoriginal rear diaphragm? One with a slightly smaller hole, or is the rear aperture on that a fixed aperture...some don't thread out.

As far as I can judge, the objective is as originally built.
Yet owing to your comment (thanks for it !), I was surprised to find found that the rear diaphragm can be removed by means of snap ring pliers, so I can now gain access to the spring. Perhaps should try again to get DF with this objective, with vs without the rear diaphragm.
By "rear diaphragm" I mean the rear end plate of the objective, not anything inside the barrel.

P.S. the iris of the rear diaphragm of the achromat 100X (which provided DF) is definitely narrower than that of the Planapo phase 100X/1.3 (which does not provide DF), for example. But then, the latter is of a much larger diameter anyway.

[patta]

In case of interest- and lacking budget for the above mentioned Nikon 1.43-1.20
my "ball" oil df condensers are still on sale...
Max objective NA revised down to 1.25.

I should warn that DF at such high NA works only for very thin and clean specimens - things thicker than like 2 micron make a mess.

[apochronaut]

DF takes place immediately once the objective N.A. falls below the minimum condenser N.A. Yet it is similar to darkness falling after sunset, when there is a period of dusk as the sun recedes from the horizon. Reducing the N..A. further overcomes reflection and ray scatter that brightens DF, Your 1.25 objective that obtains DFat a 1.2 condenser N.A, can only be understiood in terms of some restriction that is limiting the objective's N.A. well below 1.25.

[Phill Brown]

Or LWD objective with near equal na works by only viewing the illuminated subject.
Would be interested to see results of DF from an objective with a higher na than the condenser.

[apochronaut]

You get a kind if washed out BF with an odd silvery grey sheen to it. Very glary.

[patta]

It is also relatively easy to do "opposite df"
with the condenser outputting small NA (like 0.5) and a central stop patch placed inside the objective, so the direct illumination is stopped.

With this method, objective NA can be as high as will; darkfield is dark; but there are horrible diffraction artifacts.
Used as example for "dispersion staining" by McCrone
with 10x 0.25 objectives.

[Sure Squintsalot]

Success!.....(I think)

Before selling the other kidney on a dedicated Nikon oil immersion condenser, I took a few spare parts and cobbled together a working franken-condenser:

Screenshot 2023-11-22 171208.jpg (108.89 KiB) Viewed 13818 times

I took the head off an old Leitz immersion condenser head and slapped it on a useless Nikon Abbe condenser. First, after lens removal, some surgery:

Screenshot 2023-11-22 171529.jpg (160.13 KiB) Viewed 13818 times

OK...Did it work?

Screenshot 2023-11-22 171639.jpg (109.83 KiB) Viewed 13818 times

After a few hours fiddling with this thing, I was able to get reasonably bright, submicron resolution with a nice dark field using glycerin and glycerin objective. Holy cow.... I think it worked.

Screenshot 2023-11-22 171839.jpg (73.04 KiB) Viewed 13818 times

I could see details with my 100x objective in old plankton slides that I never suspected was there!

Screenshot 2023-11-22 172414.jpg (44.01 KiB) Viewed 13818 times

I don't even know what kind of immersion condenser that Leitz actually is. Anyone have any ideas?

[apochronaut]

It is unlikely to be anything but oil. In using glycerin, which isn't too far off the refractive index of immersion oil, you would be introducing a tiny amount of s.a. and altering the focal point slightly. Seems to work pretty good.
That's a nice job. DF condensers tend to be pretty neutral as to what microscope they are on. Using an orphan Nikon abbe frame as a spacer was a bright idea.

[Sure Squintsalot]

Thanks apo.

Yep, there's a tiny bit of abberation that I removed in post.

I may yet fine tune this prototype by finding the focal point of the Abbe condenser lens and setting the immersion ball-bearing lens there. The problem is that it's already so good that I'd hate to screw it up.

[Phill Brown]

Do you have a phase telescope and a slide ringing table?

[Sure Squintsalot]

I do not have a phase telescope, though it would be nice to have. I don't think I've ever seen one come up on ebay. Would a phase telescope be a useful substitute for a Bertrand-Amici lens?

....and no, I don't have a slide ringing table. Putting that much effort into a slide that may have mountant problems later on is too hearbeaking to consider. I have plankton strews mounted less than a year ago that are already showing signs of crystallization.

[Phill Brown]

If you have a spinny table and a fine tip pen you can make circles on things and check the outer ring visible with the telescope.
The 4x will show where the maximum outside NA of the condenser is.
Watch glasses are available in most diameters.
Just saying.