Maximum effective NA in darkfield - 1.0 or 1.1?

zzffnn · Post by **zzffnn** » Sun Apr 03, 2016 4:18 pm

My quick question is
what is the maximum effective/achievable NA in darkfield - is it closer NA 1.0 or 1.1?

I am using a dedicated oil condenser, with light cone NA 1.2-1.4.

I am pretty sure the highest possible effective NA lies between 0.9 to less than 1.2. My question is, is it closer to NA 1.0 or 1.1?

Thank you very much!

I know from my own experience, that NA 0.9 is achievable in darkfield, with a water immersion iris fluorite objective (set to 0.9) and Klaus Kemp test diatoms.

An AO Spencer C127 oil achromat NA 1.25 objective, stopped down by funnel stop (to around NA 0.8-0.85), produced slightly less resolution and sharpness, compared to the water immersion NA 0.9 fluorite.

I also tried a dry NA 0.95 apo objective, which made more fog/halos, but still usable and produced good resolution with test diatoms.

Such highest possible effective NA depends on subject thickness/transparency, mountant and objective immersion. Thick live ciliates in water mount cannot be images well above NA 0.75-0.8 or so, for example.

I am just asking, based on your own experience with an iris darkfield objective, what is the highest effective NA you obtained? And what was your subject type (resin mounted samples or live water protists)?

If such highest possible NA stops at NA 1.0 or less, then I have a easy and cheap solution. If it is closer to NA 1.1, then I would have to spend much more.

Ichthyophthirius · Post by **Ichthyophthirius** » Mon Apr 04, 2016 6:32 am

Hi,

I don't do much darkfield microscopy myself, but according to the manufacturers even higher NAs are achievable.

Leitz made the Heine phasecontrast/COL/darkfield condenser with NA 1.4 oil immersion front lens which was supposed to give darkfield with the

Fl 70/1.15 objective and even with the
Apo 90/1.32

but as you said, only with the very thinest objects (usually that means directly adherent to the underside of the coverslip, not free-floating) and perfectly prepared slides.

See page 6 here: microscope.database.free.fr/513_files/513-5c%20Phase%20contrast%20equipment.pdf

Regards,

Ichty

zzffnn · Post by **zzffnn** » Mon Apr 04, 2016 6:55 am

Thank you, Ichty.

Yes, I read that page 6 and saw those claims by Leitz.

The problem is I do not have a Leitz Heine condenser (it costs $400!). Mine is a regular paraboloid oil darkfield condenser with a regular light cone of NA 1.2-1.4. So for me, objective NA 1.2 is likely not possible.

As far as I know, Heine is a very special condenser, unlike most others. So what applies to Heine, probably may not work for most others.

Do you know Heine condenser's inner light cone NA, when in darkfield mode? It is over NA 1.32, I assume? How about its outer light cone NA?

Ichthyophthirius · Post by **Ichthyophthirius** » Mon Apr 04, 2016 8:03 am

Hi,

Ah, sorry, I misunderstood your question. You'd like to know the maximum NA you can achieve with your existing NA 1.4/1.2 condenser.

The light ring of the Heine condenser is VERY narrow, so the maximum setting is probably very close to 1.4 but I don't know any details.

Regards, Ichty

zzffnn · Post by **zzffnn** » Mon Apr 04, 2016 4:20 pm

And here is a somewhat related question:

I am considering two oil iris objectives (to use with an oil darkfield condenser NA 1.2-1.4), please kindly help me pick one:

1) a LOMO 60x NA 0.7-1.0 apo

I have no experience with this particular objective. But since it is a LOMO apo, and I have had all positive experiences with other LOMO apos, I am leaning towards this one.

2) a Zeiss Jena 90x NA 0.8-1.25 achromat

I returned one of those before. I suspect that particular one was defective. Test diatom image were hazy, diffused and slightly yellowish. My LOMO dry 40/0.95 apo made much better image (less hazy, sharper and no yellow color). My LOMO water immersion 30x NA 0.6-0.9, set to NA 0.9, made even better image.

The Zeiss lens itself is slightly yellowish, though clear otherwise (no delamination/fungus/scratch) and iris worked well.

I am kind of wary about that vintage Zeiss objective - unless it has obvious advantage for DF by going over NA 1.0, I probably won't buy the same model again.

I suspect in most of my darkfield microscopy, I won't be able to go above NA 1.0.

Again, most of my application is wet mount live protists. And very rarely, some prepared slides.

Thank you very much!

Pau · Post by **Pau** » Tue Apr 05, 2016 6:37 am

As we previously discussed the objective NA must be under the inner NA condenser cone, so with a 1.2/1.4 DF condenser you must stay about NA 1-1.1. The difference will not be big and when you approach to the limit things become more critical. I have no experience with that objectives, but if you're happy with lomo apos I think it could be your best option: CA is much prominent in DF.

zzffnn · Post by **zzffnn** » Tue Apr 05, 2016 7:29 am

Thank you, Pau. I agree with you.

I asked because I saw a darkfield resolution increase, going from around NA 0.7, to NA 0.8 to NA 0.9, looking at lines of Amphipleura pellucida test diatom (with a LOMO water immersion 30x NA 0.6-0.9 iris fluorite objective). NA 0.9 could resolve the lines, NA 0.8 barely and NA 0.7 failed. Thus I wonder how much better it can get, by increasing DF NA further.

Subjects and mounts surely matters. I am aware that I won't be able to go up to over NA 0.9 for many subjects and mounts (due to things like darkfield halos). It is just nice to have some margin (NA reserve under iris) just in case and know that I can cover the limits

More magnification would help too, because I can only go up to 45x NA 0.9 with K15 eyepieces. With say a 60x NA 0.7-1.0, I can go up to 90x with K15 EPs.

Charles Krebs · Post by **Charles Krebs** » Sun Apr 10, 2016 12:26 am

If such highest possible NA stops at NA 1.0 or less, then I have a easy and cheap solution. If it is closer to NA 1.1, then I would have to spend much more.

I am curious what the two options are that you refer to.

You would be very hard-pressed to see any difference between 1.0 and 1.1. I don't know how to even measure it. I use a 40/1.0 Zeiss oil lens with my Zeiss 1.2-1.4 Ultra-condenser. I also use the Olympus 100/1.40 by closing the built-in aperture until I get "clean" darkfield, but don't know exactly what NA is used.

If you have a good solution for NA 1.0 then I sure would not be inclined to spend "much more" to try to reach NA 1.1. Again, what are the two options you are looking at?

zzffnn · Post by **zzffnn** » Sun Apr 10, 2016 6:29 am

Thank you very much, Charles!

I actually have 3 iris obj choices now:

1) a LOMO 60x NA 0.7-1.0 oil apo

I have had all positive experiences with other LOMO apos. But I may be able to get close to that range, without buying that oil objective, by using my preferred watter immersion 45x NA 0.9, 60x NA 0.75 and 65x NA 0.8. Not sure about the last NA, but it is a funnel stop that we can open up slightly with lathe, in the near furture (maybe to NA 0.9?).

2) a Zeiss Jena 90x NA 0.8-1.25 oil achromat
I had one of these before and returned it due to its poor diffused/soft darkfield image. Maybe that particular objective was defective (lens was slightly yellow though perfectly clear/functional). But My LOMO dry apo 40/0.95 (fix NA) resolved better in darkfield, than that achro at any iris setting.

3) a vintage Bausch and Lomb NA 1.4 oil apo at near 100x
An experienced microscopist/dealer recommended that to me. Not sure what its lower iris limit is. The NA 1.4 would be useful for my microtomed slides. But it is rare and needs additional rare compens eyepieces and a lower power scanning parfocal objective (they won't be parfocal with my LOMOs on my nosepiece).

Your Zeiss 40/1.0 has lower iris limit of 0.6, correct? Have you ever used your Zeiss at full NA 1.0 in darkfield for anything?

I am not sure if NA go up linearly with iris turning ring, if it does, then we can guess the middling NA?

I actually think having an iris at above NA 0.9 may be useful for darkfield now, after yesterday's experiment. Even with live water mount, some tiny diatom-sized flat subjectives do benefit from using NA 0.9 darkfield. Though it seems NA 0.9 is not far from the resolution limit of darkfield, since it resolved lines of test diatom Amphipleura pellucida for me (in DF).

Pau · Post by **Pau** » Sun Apr 10, 2016 7:40 am

I haven't any iris bundled objective. For high NA dark field my choice is the Leitz NPL Fluotar 50/1.00 oel 160/0.17. It's a really nice DIN objective with less CA than other fluorites, in fact in practical terms it behaves like a Plan Apo.

zzffnn · Post by **zzffnn** » Sun Apr 10, 2016 8:10 am

Thank you, Pau. That is a good data point for me! I have heard many praises for Leitz objectives.

zzffnn · Post by **zzffnn** » Sat Apr 16, 2016 11:05 am

I will answer this question myself with results from an experiment:

NA 1.1 darkfield is possible, with 1.2/1.4 oil darkfield condenser oiled, a mounted diatom slide and NA 1.1 65x LOMO water immersion objective (immersed in water above cover slip).

I pushed it with 15x to make it to 97.5x NA 1.1 darkfield. My 40w LED barely provide enough light at that magnification/NA. NA 1.1 actually helped by gathering more light.

I probably forgot to try NA 1.1 darkfield previously, or I failed to obtain good optical alignment.

Of course, this is only good with tiny diatoms. A thick and big ciliates may not work at NA 1.1 darkfield, of course.

We will make some funnel stops of different NA (0.85 and 0.95) next for the 65x objective. We already have NA 0.65 and 0.75 at 40x and 60x. NA 0.65-0.9 at 30x and 45x.