Illumination techniques for beginners

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sreynolds
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Illumination techniques for beginners

#1 Post by sreynolds » Wed Mar 22, 2023 1:37 am

Learning about my microscope. Biological Labophot with Abbe condenser and 3D printed filter adapter for condenser to take darkfield patch stops and filters. Labophot has Nikon analyzer under the head. My intended subject material is aquatic organisms, plant and animal. Need to know when to use what setup. The subject here is cotton fibers from a Q-tip swab. They are birefringent, so good material for showing polarized light use. Objective is Nikon E Plan 10x, and camera is direct projection onto Olympus E-PL7 micro 4/3.
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My understanding is that a retarder, if used, should in theory be placed between the objective and the analyzer, but there is no place on my Labophot to do that unless I stick it under the analyzer, where is is inconvenient to get at. I don't want to take the head off and on a lot. So, in my case the retarder goes in the 3D printed adapter in the condenser, above the polarizer and below the stage (obviously).
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I haven't had good luck with darkfield at 40x (or above), and found darkfield can generate a lot of CA on bigger objects because of being lit up from the side, so for now the most useful setup seems to be the polarizer with the retarder plate. I've tried a mica retarder, and this 1/4 wave one which certainly gives nicer colors than the mica.
Steve

Hobbyst46
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Re: Illumination techniques for beginners

#2 Post by Hobbyst46 » Wed Mar 22, 2023 12:34 pm

Nikon E-Plans produce very good images, at least in brightfield as per my experience.
Without a dedicated darkfield condenser, many fail to achieve DF with an NA 0.6-0.7 objective.

Sure Squintsalot
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Re: Illumination techniques for beginners

#3 Post by Sure Squintsalot » Wed Mar 22, 2023 7:35 pm

On my petrographic set-up, the waveplates slide in above the objective and under the analyzer. That's where it belongs for performing any quantitative microscopy, I believe. If you're just looking to create interesting lighting effects, by all means put it anywhere it fits! Some of that stuff can look pretty cool.


For anyone that knows anything about darkfield, are high N.A. objectives incompatible with achieving darkfield effects? I have a 20X fluor objective with a NA of 0.75 and I cannot make it work. An ancient Leitz 25x objective with a NA 0.45 works well enough.

dtsh
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Re: Illumination techniques for beginners

#4 Post by dtsh » Wed Mar 22, 2023 8:33 pm

Sure Squintsalot wrote:
Wed Mar 22, 2023 7:35 pm
On my petrographic set-up, the waveplates slide in above the objective and under the analyzer. That's where it belongs for performing any quantitative microscopy, I believe. If you're just looking to create interesting lighting effects, by all means put it anywhere it fits! Some of that stuff can look pretty cool.


For anyone that knows anything about darkfield, are high N.A. objectives incompatible with achieving darkfield effects? I have a 20X fluor objective with a NA of 0.75 and I cannot make it work. An ancient Leitz 25x objective with a NA 0.45 works well enough.
I'm no expert in darkfield, but my guess would be the 20x might need to have it's NA stopped down somehow based on my limited experience with AO's darkfield where using the 100x objectives requires installing a funnel stop which reduces the NA.

PeteM
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Re: Illumination techniques for beginners

#5 Post by PeteM » Thu Mar 23, 2023 12:55 am

Simple darkfield stops usually work fine up to around 20x objectives, assuming they are not higher numerical aperture fluorites or apochromats. You can often get a semi-dark effect up to 40x by fiddling with the condenser centering and height - but it's usually not highly effective. Above that, you need a dedicated darkfield condenser with its own numerical aperture higher than whatever objective you plan to use.

Many systems offer two different dedicated darkfield condensers. One dry, for 40x or so. Another for oil immersion (both the objective to the slide and the slide to the condenser) for use at 50-100x. In most cases, that oil immersion objective will require an iris or a stop to reduce its numerical aperture.

It's all pretty much a matter of geometry. High numerical aperture objectives "see" a wide-angle view. Lower numerical aperture condensers project a narrower view (or ring of light in the case of darkfield), thus destroying the darkfield effect.

In response to the original question, simple polarization can sometimes add useful contrast for pond critters. Darkfield can be wonderful for many subjects, but it's fussy and will show artifacts. Oblique is somewhat similar, but worth trying. DIC probably reigns for small aquatic creatures but is expensive. Phase contrast is more affordable and effective, though at the expense of "halos" and a slight loss of resolution.

apochronaut
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Re: Illumination techniques for beginners

#6 Post by apochronaut » Thu Mar 23, 2023 3:02 am

Each illumination system has unique capabilities and reveals information that others don't. Profiling one technique as superior to another : obsessing over DIC for instance , just shows a lack of experience in microscopy.
Irregardless of the technique, critical adjustment of the entire system and especially related to centering is the key to optimizing all illumination techniques. It may come as a surprise to many that phase contrast for instance lacks 3 dimensionality when only a fraction out of longitudinal and lateral adjustment perfection but does have some, if perfectly centered and perfectly adjusted longitudinally. Yet, it can also sometimes benefit from being combined with oblique, off center.
There are two mantras when it comes to illumination. Center and Offcenter. Learning when to use each and when to combine them is the key to being illuminated about illumination.

Sure Squintsalot
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Re: Illumination techniques for beginners

#7 Post by Sure Squintsalot » Thu Mar 23, 2023 3:43 am

PeteM wrote:
Thu Mar 23, 2023 12:55 am
High numerical aperture objectives "see" a wide-angle view. Lower numerical aperture condensers project a narrower view (or ring of light in the case of darkfield), thus destroying the darkfield effect.
I think that's the key: what the condenser projects is not matched by the viewing angle of the high NA objective. I just found a neat applet on the Olympus site effectively explaining it. That 20x Fluor objective is a pretty nice one but now it seems I have to find a specific condenser that will allow DF. More Gear!

apochronaut
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Re: Illumination techniques for beginners

#8 Post by apochronaut » Thu Mar 23, 2023 11:10 am

Sure Squintsalot wrote:
Thu Mar 23, 2023 3:43 am
PeteM wrote:
Thu Mar 23, 2023 12:55 am
High numerical aperture objectives "see" a wide-angle view. Lower numerical aperture condensers project a narrower view (or ring of light in the case of darkfield), thus destroying the darkfield effect.
I think that's the key: what the condenser projects is not matched by the viewing angle of the high NA objective. I just found a neat applet on the Olympus site effectively explaining it. That 20x Fluor objective is a pretty nice one but now it seems I have to find a specific condenser that will allow DF. More Gear!
The principle of DF is very simple. It is a modified form of circular oblique lighting ( COL) that results in total internal reflection in the slide itself, (TIR). There are 2 conditions for successful DF illumination .
1) Illumination by a source that is at least .20 N.A. above the N.A. of the objective in order to eliminate flare and scatter.
2) Completely uniform symmetrical illumination, therefore precise centering. DF condensers do most of that for you, whereas with patch stops at higher N.A's you might as well take up plate balancing and go on ( fill in your country of choice)'s Got Talent.

DF condensers have a minimum and maximum N.A., which creates a ring of illumination, like a thin doughnut. It's not really a cone, it is more like the walls of a funnel, projecting it's illuminated cross section on the slide. If the objective can't see it, then theoretically you can have DF.
This N.A. range of that doughnut is often stamped on the condenser but if not, a slide thickness specification may be, which is a related specification. The maximum N.A. is usually about .20 higher than the minimum, so since any dry DF condenser cannot have a maximum N.A. higher than 1, therefore it's minimum will be approximately .80 to .85 , maybe .90 is possible but that would be a really precise tightly engineered condenser and would require high illumination. Never seen one like that. This is where it gets a bit confusing because technically if the objective cannot see the projected ring and therefore the minimum condenser N.A. is higher than the objective, there should be DF. That gets stated a lot in print and by people who have never actually done DF, just read about it. In practice, when the objective N.A. is too close to the lower condenser N.A., a whole group of elements from glass impurities to sample refraction and extraneous reflection cause illumination scatter, turning DF into grayfield, which is kind of like dawn. In order to get really good DF with a good dark background you need to have the condenser's minimum N.A. .20 above the objective at least to be safe, which means an oil DF condenser, for an objective of .75.
This is evidenced by the Reichert Univar microscope and maybe the Polyvar too. They both had the advantage of a really good DF condenser that had a quite wide illumination funnel or ring between 1.40 and 1.20 N.A., so very high. Since all of the objectives for those microscopes were entirely universal and used for all contrast methods, they all had to be D.F. ready. They made every objective with an N.A. above .75 with a built in iris diaphragm. 3- 40X, 2-63X and 4- 100X. So, .75 seems to be an accepted cuttoff point, since they made a 25X .65 planapo which works stellar with their oil condenser and did not come fitted with an iris. For the 10 or so objectives they made with N.A.s below .50, they also fitted s dry DF condenser into the substage.

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