Judging high-NA infinity-corrected objectives

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Lou Jost
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Judging high-NA infinity-corrected objectives

Post by Lou Jost »

With the recent glut of 20x 0.75 Nikon objectives, it might be worth reminding people of how special it is to have high NA in a low-power objective.

The resolving power of an objective on the subject depends only on the NA, not the magnification of the objective. The standard 200mm tube focal length is not optically anything special, and any other tube focal length will still preserve the resolution on the subject. The lower power infinity-corrected objectives differ from higher-power infinity-corrected objectives mainly in their bigger image circles at any give tube focal length.

That means you can use the 20x 0.75 objective at 40x or even more, using a 400mm tube lens, with no degradation relative to a purpose-built 40x 0.75 used with a 200mm tube lens. This 20x pushed to 40x should outperform 40x 0.65 objectives.

So for a given NA, a lower-power infinity-corrected objective is much more useful than a higher-power one. You can use it at the lower magnification and you can also use it at higher m without facing empty magnification..

By the way, a good 400mm tube lens is the old manual Nikon ED-IF f/5.6 lens.

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Post by ChrisR »

Indeed. I was just today reading about an Oly 10x NA 0.6, and reflecting on how the NAs they are a changing.

I twitched at "depends only on the NA.... of the objective".
I'm not a microscopist. One day, maybe. I have read about Herr Köhler; we need to illuminate with at least the same width of light-cone, I understand, otherwise we lose resolution. OK.
(But the August gentleman also said one must focus the filament at the back focal plane of the objective. Is this only to ensure it's completely out of focus? If so I'm not 100% sure why it has to be just there.)

I am wandering from Lou's subject, but to bring it back, If I use a 20x NA0.75 objective on a camera/tubes setup, I have to illuminate the subject from beneath.
Can I simply use a diffused large light source (starting, say, with a kitchen cupboard underlight),
Image
sit a condenser right on it, and align the axis with the objective?
It needs height adjustment of course. For a "field diaphragm" can I use a hole in black paper?

What would I, or any other prospective lens tester, be missing?
Chris R

Lou Jost
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Post by Lou Jost »

Chris, When I said the resolution depends only on the NA and not the magnification, I was only talking about the objective's side of things. The resolution also depends on all the other usual things outside the objective.

For transmitted light, such as my test of the 20x on another thread, I used a simple thing inspired by an LED "condenser" that was recently published. That was a hemisphere of LEDS and microlenses focused on the subject. The subject sits on a glass slide atop the upward-facing hemisphere. My DIY version is half a ping-pong ball glued to the bottom of a cover slip resting on a glass of dilute milk, so that the shell of the hemisphere is almost completely immersed in the milk (but is dry on the inside). The inside of the glass is spray-painted matte white except for a few clear areas for beams of light to enter. The milk thus glows uniformly when illuminated through the clear windows, and illuminates the half-ping-pong ball, which then provides perfectly even illumination through the subject. The milk diffuser is essential for when I work with monochromatic lasers, the best light for high resolution work since there is no absolutely zero CA. The lasers need to be de-speckled, and dilute milk does that.

But there are other alternatives, like epi lighting. Though this could be hard for objectives that require a cover slip, because of reflections. BD objectives are also an option.

But I should stress that my post was just about objectives. Lighting for the 20x 0.75 is no harder than lighting for a 40x 0.75; my main point is that a 20x 0.75 can be used in any situation where a 40x 0.75 could be used.

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Post by ChrisR »

I wasn't doubting anything you said Lou, just reacting to it - one thought spawns another, and a set of questions.

Do you use a field diaphragm with your milk and hemispere arrangement?

I wonder id you've used a less smelly/ temporary scattering mix, such as latex paint or tapioca powder in gel?!
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Lou Jost
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Post by Lou Jost »

Chris, no, there is no real diaphragm but there is a black paper mask on top of the subject, with a small hole in it, to keep stray light from entering the lens. That serves the same function.

I would love to find a non-organic substitute for milk. The point is not just to diffuse the light, but also to de-speckle it. For that to happen, the diffusing particles have to be moving around at a good rate, so that the interference patterns average out during an exposure. I don't think a gel could work. Maybe some kinds of paint would work, but milk is the traditional de-speckling medium among laser physicists (I used to be one many years ago).

By the way, it would be fine to doubt what I say!! Doubt is a good thing, and I can always be wrong. The only way I 'll find out if I am wrong is if someone doubts me.

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Post by ChrisR »

I thought latex paint de-speckled - Mie scattering. But I'm sure you're way ahead of me!
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Lou Jost
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Post by Lou Jost »

I can't say I have tried it. It is worth a try. Milk does get stinky after a few days. But paint might leave a stain? Milk doesn't.
Last edited by Lou Jost on Mon Dec 31, 2018 8:37 pm, edited 1 time in total.

abednego1995
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Post by abednego1995 »

Has anyone used a single mode optic fiber for scrambling lasers? I've only read descriptions in text, and it seems simple enough. Get a fairly long fiber coupled with a laser, make a few simple loops in it... and vibrate the loop at some frequency, and voila, you get despeckled light out.

It should lose less light than scattering, and depending on the exit angle from the fiber, should become a nice ultra bright point source which is important in microscopy.

Lou Jost
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Post by Lou Jost »

I haven't tried that, thanks for the suggestion.


However, I do like the diffusing action of milk. The diffusion is much more complete than the things we usually use to diffuse light in macro photography.

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Post by Pau »

Lou, could you kindly post pictures of your "milk condenser" to fully understand it?
Pau

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Re: Judging high-NA infinity-corrected objectives

Post by Pau »

Lou Jost wrote:With the recent glut of 20x 0.75 Nikon objectives, it might be worth reminding people of how special it is to have high NA in a low-power objective.

The resolving power of an objective on the subject depends only on the NA, not the magnification of the objective. The standard 200mm tube focal length is not optically anything special, and any other tube focal length will still preserve the resolution on the subject. The lower power infinity-corrected objectives differ from higher-power infinity-corrected objectives mainly in their bigger image circles at any give tube focal length.

That means you can use the 20x 0.75 objective at 40x or even more, using a 400mm tube lens, with no degradation relative to a purpose-built 40x 0.75 used with a 200mm tube lens. This 20x pushed to 40x should outperform 40x 0.65 objectives.

So for a given NA, a lower-power infinity-corrected objective is much more useful than a higher-power one. You can use it at the lower magnification and you can also use it at higher m without facing empty magnification..
I agree, of course.
I want just to add that the rise of high NA objectives is in a big part driven by the needs of modern fluorescence techniques, where most big advancements have been done in optical microscopy in the last few decades.
Light gathering increases with the square of the NA and epifluorescence intensity with the fourth power of it, so high NA is a key factor for detecting weak fluorescence.
Nowadays we can see those beautiful high NA plan apos but time ago it was often only offered with non plan fluorites.
Pau

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Post by Lou Jost »

Pau, here is the top part. The subject sits on top of the cover slips that cross the hemisphere. The extra glass is to provide structural strength so the cover slips don't break. I made this before I knew how to cut glass, so I had to break the reinforcing glass strips with a hammer, hence the sloppiness.

Image
Image

Here is the cup that the hemisphere assembly sits on. The cup is to be almost filled with dilute milk (leave room for the displacement of the hemisphere!). Lasers shine into the milk through that transparent band. The glowing milk lights the ping pong ball uniformly, and this in turn illuminates the subject uniformly from all directions below.

Image

The undersides of the cover slips fog up if the milk gets hot from the light, so if I did this again I would leave out one of the cover slips, to provide ventilation to the inside of the hemisphere.

Scarodactyl
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Post by Scarodactyl »

On the one hand, the thick cover slip correction could make these somewhat inconvenient. On the other hand, I wonder if it would be an advantage for viewing mineral inclusions inside gems. The RI of most gem materials will be a lot higher than glass, but definitely closer to glass than to air.

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Post by Pau »

Lou, interesting and very unique invention!

If I understand it well, the hemisphere is filled with air, isn't it?

I don't doubt that you're getting uniform illumination with the device but I'm not sure that it will fulfill the rear aperture of a high NA objective.
As you know for sure, the goal is that all rays of light emerging from each point of the subject will enter the front aperture of the objective and fulfill the rear aperture of it (correct me if wrong, pls)
In your device the actual source illuminating the subject is placed pretty far from it. I fear that this will limit the illumination angle, not sure.

An illuminated diffusing surface placed just under the slide would be more effective for the task -like in the Zeiss Lumigen condenser or in the Chris LED plate proposal
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Post by Lou Jost »

Pau, yes, the hemisphere is filled with air.

As for the theory, I am also not sure, I hope Rik can illuminate the issue. I will do some experiments with it. A simple flat milk diffuser (the cup filled with dilute milk but without the hemisphere) also works well at low m. I will try to compare them at high NA one of these days.

I do need the milk rather than an LED so I can take advantage of despeckled laser light.

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