Bright field condenser usage for high NA and Koehler illumination

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Bright field condenser usage for high NA and Koehler illumination

#1 Post by kit1980 » Tue Jul 26, 2016 1:34 am

I've read today in different places (for example, http://www.olympusmicro.com/primer/anat ... nsers.html), that even if a bright field condenser is marked 1.25 NA, without oil it will be max 0.95 NA. So, to use with high NA 100x objective the condenser needs to be oiled to the slide.

To be oiled to the slide, the condenser needs to be very close to the slide. On the other hand, for Koehler illumination the condenser needs to be at some greater distance from the slide. How to solve this dilemma?
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Re: Bright field condenser usage for high NA and Koehler illumination

#2 Post by apatientspider » Tue Jul 26, 2016 5:10 am

To be oiled to the slide, the condenser needs to be very close to the slide. On the other hand, for Koehler illumination the condenser needs to be at some greater distance from the slide. How to solve this dilemma?
If the condenser is properly matched for your illumination system, it should be close enough to the slide for oil contact even when focused for Kohler illumination. You should keep in mind that some condensers have longer or shorter focal points than others. Also, some condensers - especially older ones intended for use with a mirror and lamp - are designed to accept light rays from infinity, whereas others are designed to accept them from a much closer source.

For example: My scopes (Spencers) all use a substage condenser, mirror and separate lamp with its own condenser for illumination. The lamp iris is supposed to be positioned 8 to 10 inches away from the aperture or iris of the substage condenser. Normally I have no trouble getting the various substage condensers to focus close to the bottom of the slide when setting up for Kohler. However, some time ago I purchased an American Optical aspheric condenser to try on one of them. It was intended for an AO model 10, which will do Kohler but has a built-in illumination system. The condenser fit the yoke of my particular microscope - a Spencer model 15 - but would not focus the lamp iris in the object plane on top of the slide when using a separate lamp. The focal point was somewhere inside the slide - even a very thin one. So it was just the reverse of your difficulty.

By subsequent experimentation I discovered that the closer I moved the lamp iris to the mirror and aperture of the substage condenser, the higher up the condenser would focus. When I got the lamp iris close enough - about where the illuminator of a model 10 is, right under the stage - the aspheric condenser would focus the lamp iris at he proper place for Kohler, ie., in the object plane. A bit later on I bought an illuminator for a model 10 off Ebay and tried it under my model 15 with the aspheric condenser (had to raise the scope on some blocks to get the illuminator to fit.) Everything focused perfectly fine!

But as I said, you seem to have just the opposite problem - your condenser sits too far away when focused for Kohler. Is it the one that was intended for your microscope? Someone here had the same problem a while back. He had been sold a different condenser than what normally was supplied with his model scope so that he could do Kohler illumination or darkfield or something, and then found he could not bring it close enough to the bottom of the slide for oil contact. Not sure what he ended up doing about it.

Someone else in this forum suggested the following, but I've also read it in some old book (memory fails.) If you wish, you can try oiling and stacking some small round cover slips or pieces of specimen slide to the top element of the condenser to take up the space, depending on how much space you need to fill. You can stack the pieces together temporarily with immersion oil ( rather messy) or you could glue them permanently with balsam to make one solid spacer, and then oil it to the slide and the condenser. If you need to do so, specimen slides are easily cut with heavy scissors or tin snips fully submerged in a pail of water - glass, snips, hands, everything completely under the water.

Alternatively you might try using a thicker specimen slide - or try stacking a couple together with oil. That would require the condenser to be raised higher to focus properly.

Another, easier and less messy solution would be to obtain the proper spectacle lens to place between the illumination source and the iris of your substage condenser to bring the focal point to the proper place. What you'd want is something that would make your illumination source seem farther away. Someone has suggested a positive lens from a pair of non-prescription reading glasses - "cheaters" we call them here in the States.



Actually though, most of the experts here will tell you the question is rather moot anyway. A condenser with an n.a. of 1.25 is most likely just an uncorrected, two element Abbe. Even oiled you would need quite educated eyes to see any appreciable difference over using it dry, because the total numerical aperture of your system is going to be somewhat less than 1.25 with an uncorrected condenser - even oiled. You'd be better off to obtain a usable, fully corrected condenser of n.a. 0.95 and use it dry. Even just an aspheric or aplanatic condenser used dry would be better than an Abbe - especially if used with a green filter.

Jim

Edit: See Gekko's post about a positive camera lens in this thread and where to obtain them. viewtopic.php?f=18&t=2720 The OP seems to have had the same problem as you.

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Re: Bright field condenser usage for high NA and Koehler illumination

#3 Post by gekko » Tue Jul 26, 2016 12:07 pm

I agree with apatientspider: if the condenser was meant for your microscope, then it should be close enough when you have Koehler illumination correctly adjusted.

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Re: Bright field condenser usage for high NA and Koehler illumination

#4 Post by apochronaut » Tue Jul 26, 2016 1:53 pm

Jim offers a number of interesting points about condensers, something I have fiddled with a lot, in order to optimize imaging. The consumer marketplace woos us easily with pictures of inexpensive, nicely modern microscope bodies, serious looking objectives and all the right words in order to convince us, that for the price asked, there can be no finer purchase than the one you are looking at in the picture. Almost always the words "kohler illumination" or "illumination by kohler" is part of the package. Why, it's almost as though " body by Fisher" has been reincarnated, minus the rust, although as Neil Young pointed out, rust never sleeps. I 'd like to add that it just morphs into something else. In this case, it's chromatic and spherical aberration.
Jim has pointed out that there are two types of condensers, that otherwise can look similar. I would also add that one of them is finitely corrected and the other is infinity corrected. Essentially, although the focal point is different for the two due to the distance from the illumination source, each one is the reverse of the other. To start, it is important to know that the bottom lens of a condenser is the front lens.
Condensers, designed for critical illumination are focused on infinity. They receive a bundle of parallel rays from an illuminator collector lens, or as in the case of remote illuminators, from a distance, and focus the illumination beam convergent in the object plane. The converging rays create a degree of scattering in the slide but in a well set up system , this can be made minimal. Those that are designed for kohler illumination are focused at a finite distance, the distance between the condenser aperture plane and the field iris plane, they then convert that diverging beam into a parallel beam at the image plane. If the condenser must be lowered, as is the case in this thread, in order to achieve what appears to be kohler illumination, it does imply, that possibly it is a condenser designed for critical illumination, or the microscope has not been designed for kohler illumination in the first place.
This isn't so bad. There is a lot of talk about kohler illumination, as though a microscope is malfunctioning without it. Probably half or more of the major microscopic work done over the last 1 1/2 centuries was done without kohler illumination and here is why. Up to apertures around .45, the illumination beam has little effect on resolution. In fact you can take the condenser right out of your microscope and achieve, maybe not as bright but certainly as sharp imaging. The primary job of a condenser is to maximize resolution, by providing a light beam that equals the optimum N.A. of the objective.
Most average kohler systems, use a cheap abbe oil condenser of between 1.15 and 1.3 N.A., or usually 1.25, which seldom work above .90 dry. Abbe condensers have loads of lateral chromatic and spherical aberration. At low N.A.'s, those defects aren't a factor but as the N.A. goes up, requiring the full aperture of the condenser to achieve good resolution, those two defects become readily apparent and the aperture needs to be kept stopped down in order to reduce them and provide better contrast. So, with kohler illumination and an abbe condenser, it is really impossible to achieve the resolution that the objective is capable of. In fact, at high apertures, critical illumination provides better imaging than kohler. The problem associated with the lamp filament is easily overcome by diffusion filters( usually included in the collector lens system) and slight defocusing of the lamp.
I also agree with Jim, with regards to oiling the condenser. For all around general purpose use, a high N.A. achromat condenser is the best and easiest to use. Abbe aspheric condensers are almost as good dry and about as good when oiled. When it comes to high N.A. achromat condensers, they really need to be oiled, to make their purchase worthwhile and they are definitely superior, in all ways except convenience and price. The ones I have tested, even with very high theoretical N.A.'s( 1.4) when oiled, performed less well when used dry, than a .90 dry achromat.
I do think, however, if you are needing to get every last bit of potential resolution out of an objective with an N.A. above 1.0, oiling even an abbe condenser, despite it's messiness ( one gets pretty quick and adept at cleanup, once you've done it a bit) is worth it but I also agree that eventually, acquiring an aspheric or or dry achromat condenser, is the best solution. The ultimate N.A. of an optical system is a combination of the objective's N.A. and the condenser's N.A., if lower than the objective. A dry abbe condenser working at .90, used with an immersed objective at 1.25 will have a working N.A. of about 1.10-1.15 but if the field aperture or condenser aperture needs to be closed, in order to overcome defects in the condensers performance, such as spherical aberration, glare or chromatic aberration, the resulting N.A. could be much lower. OIling the condenser, allows for a fuller use of the available aperture.
A mention needs to be made about the collector lens in a microscope. If installed, they sit between the illuminator and the condenser. They are either focused for Kohler capability or not. Usually, in order to even out the filament image the collector lens pack in a non-kohler system includes a diffusion filter. There would be less need for a kohler type collector lens to include that feature. A kohler type collector lens converts a diverging beam into a converging beam. A non-kohler type collector lens, converts a diverging beam into a parallel beam.

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Re: Bright field condenser usage for high NA and Koehler illumination

#5 Post by mrsonchus » Tue Jul 26, 2016 2:01 pm

Gentlemen, this is a very interesting thread indeed, from which I'm learning an awful lot - thanks all for the detailed in-depth and very illuminating discussion - very valuable information indeed here.
:)
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Re: Bright field condenser usage for high NA and Koehler illumination

#6 Post by kit1980 » Tue Jul 26, 2016 4:23 pm

Thank you all!

I have a Chinese microscope, and the condenser came with the scope, but it's probably designed for critical illumination, because "Kohler Illuminator" that came with it is an optional item to put over the illuminator (LED).

Anyway, I decided to use it non-oiled for now, and maybe try to buy a better condenser later.
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Re: Bright field condenser usage for high NA and Koehler illumination

#7 Post by apatientspider » Wed Jul 27, 2016 7:35 am

kit1980 wrote:Thank you all!

I have a Chinese microscope, and the condenser came with the scope, but it's probably designed for critical illumination, because "Kohler Illuminator" that came with it is an optional item to put over the illuminator (LED).

Anyway, I decided to use it non-oiled for now, and maybe try to buy a better condenser later.

Do you happen to have a photo of your scope or is there one online somewhere? It would be interesting to see this "kohler illuminator". I'm guessing it is a lens that modifies the light coming from the LED in some way, but I'd like to know exactly in what way. Does the LED illuminator have any other lenses?

An easy way to tell if your substage condenser is designed for focusing parallel rays, or light from infinity, is to substitute - if possible - a mirror and a distant source for the built-in illumination. Just focus a low power objective (the 10x) on a slide. Then use a mirror under the substage condenser - if there is room - to catch and reflect an image of a tree or something from outside a sunlit window. Without changing the focus of the objective, use the condenser to focus the image of the tree in the object plane. If it will focus a sharp image, then it is designed for parallel rays.

Also, to add to what Apochronaut wrote about condensers, there is a book I can recommend by John Belling titled The Use Of The Microscope. It contains several good chapters on the merits of various condensers and illumination techniques as well as many of the other finer points of microscopy. I'm afraid it is rather dry reading, since it contains almost no photos and few illustrations, but it is extensive. It was published in 1930, but the principles are still relevant - and it is available online to read or to download free in pdf form and other formats at https://archive.org/details/useofmicroscopeh00bell

Jim

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Re: Bright field condenser usage for high NA and Koehler illumination

#8 Post by kit1980 » Wed Jul 27, 2016 8:02 am

apatientspider wrote: Do you happen to have a photo of your scope or is there one online somewhere? It would be interesting to see this "kohler illuminator". I'm guessing it is a lens that modifies the light coming from the LED in some way, but I'd like to know exactly in what way. Does the LED illuminator have any other lenses?
I bought this microscope https://www.amazon.com/gp/product/B00UCEPH70, the page has detailed photos.

This "kohler illuminator" is just a field diaphragm. It doesn't have any lenses (but has some plain glass). The LED illuminator has lenses.
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Re: Bright field condenser usage for high NA and Koehler illumination

#9 Post by apatientspider » Wed Jul 27, 2016 8:36 am

Ha! Another night owl!

I went and took a look at your link. That's really a fairly decent-looking microscope. It takes more than just the addition of a field iris to give you kohler illumination, though - you'd have to replace or change the focus of the lenses in the illuminator. Even so, the kohler iris is a nice addition to have even if you were to set up for critical or Nelsonian illumination, because it helps control glare. In that case though, it becomes another aperture iris - like the substage condenser iris.

Another advantage of that kohler iris is this: If you wanted to work without the substage condenser and its attached iris - which you might want to do with lower power objectives - you would still need an aperture iris or diaphragm of some sort. The kohler iris would fill that need.

Jim

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Re: Bright field condenser usage for high NA and Koehler illumination

#10 Post by apatientspider » Wed Jul 27, 2016 9:18 am

Something else occurs to me: Have you ever tried focusing the substage condenser for critical illumination? That is, have you tried focusing an image of the LED at the object plane of your scope so that the specimen and LED itself are in focus simultaneously? Try that and see if the top element of your condenser doesn't reach high enough to oil to the bottom of the slide. A condenser can be oiled whether it is used for either critical or kohler illumination.

As Apochronaut writes above, microscopists used critical illumination for many years before the widespread acceptance of Kohler illumination about fifty or sixty years ago. The main reason Kohler won out was because it gets rid of the image of the bulb filament and also provides more even lighting for photography. At one time there were made various fancy illuminators and bulbs for use with critical illumination which solved the problem of the image of a coiled filament in the field of view, because they didn't use one. Some used a metallic plate for a filament, and others used a miniature electric arc. Point O Light bulbs were an example of the latter. Unfortunately they stopped being made many years ago.

Now we have LEDs available, which should solve the problem of uneven lighting caused by a coiled bulb filament. Seems to me they would be ideal for critical or Nelsonian illumination.


Jim

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Re: Bright field condenser usage for high NA and Koehler illumination

#11 Post by 75RR » Wed Jul 27, 2016 10:19 am

Try this tutorial - it should help with setting up the right condenser height:

http://zeiss-campus.magnet.fsu.edu/tuto ... flash.html

Tip: Start with aligning the filament
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Re: Bright field condenser usage for high NA and Koehler illumination

#12 Post by mrsonchus » Wed Jul 27, 2016 11:49 am

That's three times I've printed-to-PDF this thread now - so much good information being proffered - wonderfully useful and informative chaps. :)
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Re: Bright field condenser usage for high NA and Koehler illumination

#13 Post by apatientspider » Wed Jul 27, 2016 12:12 pm

mrsonchus wrote:That's three times I've printed-to-PDF this thread now - so much good information being proffered - wonderfully useful and informative chaps. :)

Well, John B., for my part, thank-you very much. It's always nice to have one's efforts appreciated.

Btw, I visited your new website. Congratulations!

Knowing your interest in botanical work, I think you might find it rewarding to peruse the book by Belling that I recommend above - if you can find the time. As I understand it, he was quite a cyto-geneticist who developed the iron-acetocarmine staining technique for detailed viewing of chromosomal structures. Mostly in plants, I believe.

Regards,

Jim

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Re: Bright field condenser usage for high NA and Koehler illumination

#14 Post by apochronaut » Wed Jul 27, 2016 1:39 pm

Yes..... hope this helps in solving condenser speedbumps for anyone encountering them.

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