Dark field illumination: Questions and answers

Here you can discuss different microscopic techniques and illumination methods, such as Brightfield, Darkfield, Phase Contrast, DIC, Oblique illumination, etc.
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Micro-Bob
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Dark field illumination: Questions and answers

#1 Post by Micro-Bob » Thu Mar 01, 2018 11:07 am

Hello together,

I opened this thread to continue the dark field discussion from the thread viewtopic.php?f=28&t=5751.

ebay Germany: I think there is just one ebay but you reach it over different URLs. You can search US-Ebay from german ebay and vice versa.
The markets for used microscope stuff are probably very different from country to country so it may be a good idea to buy rare stuff from somewhere else.

@Hobbyst46: You have a very nice and versatile condenser there! It shurely would be costly and timeconsuming to hunt one down on the used market. In contrast to a real darkfield condenser it will be only so-so in darkfield use. The cardioid darkfield condensers make it possible to have a narrower light cone and thus use a higher objective n.a. The cut off between light cone and darkness is sharper here. I will have a look at home for a dark field funnel stop. I think they were just put into the objective from the back. I guess that darkfield was not as popular from the 50s on because phase contrast was becoming more popular. I think in practical application phase contrast was more or less replacing dark field.
The distance Apochronaut mentioned: I think your microscope has a frosted lens before the mirror in the base. So probably the distance between frosted lens and lamp has not much influence on the illumination, except for the brightness.
The DIY darkfield stops: I have played aroung quite a bit with different stops and filters in different condensors. I found that with some condensors and lighting setups this is much easier than with others.

@Apochronaut: Thank you for sharing you experience! It looks as if the components cardioid condenser, dark field stop, objective aperture and funnel stop were used for acheiving dark field. For dark field stops I know only of applications up to max. a 40:1 objective. Cardiod condensers have apertures bands from ca. 1 to 1,4 but there were also weaker versions. I know of oil immersion objectives 90:1 0,8-1,25 and 60 0,6-1,0. Which combinations have been suggested by the different makers? Was there a combination of cardioid condenser and funnel stop together? I have to have a closer look: I have an english Baker? objective 80:1 Apo n.a. 1,32 that was intended to be used with a funnel stop iirc. I think the description is enclosed and will post here what they suggest.

My 90:1 0,8 - 1,25 achromat is an old pre war Zeiss design with quite heavy field curvature. With a funnel stop I could use more modern objectives for dark field.

Bob

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Re: Dark field illumination: Questions and answers

#2 Post by Hobbyst46 » Thu Mar 01, 2018 12:55 pm

Hello Bob
Thanks for opening the new post and for the as-always help and good will.
I think your microscope has a frosted lens before the mirror in the base. So probably the distance between frosted lens and lamp has not much influence on the illumination, except for the brightness.
Yes, indeed there is a frosted lens, and naturally, brightness decays with with squared distance.

Surfing, I found this fine source:
"Chapter 9 Dark Field Microscopy © C. Robert Bagnell, Jr., Ph.D., 2012"

Description and explanation of DF and specific DF condensers, funnel stops etc.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

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wporter
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Re: Dark field illumination: Questions and answers

#3 Post by wporter » Thu Mar 01, 2018 3:42 pm

Actually, in a collimated beam, the brightness will not fall off that much (as the sq. of the distance), which is exactly why they sometimes have a concave mirror behind the light bulb, and the adjustable condenser-like collector lens after it. Keeps the light from spreading out!

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Re: Dark field illumination: Questions and answers

#4 Post by apochronaut » Thu Mar 01, 2018 4:28 pm

Micro-Bob wrote: @Apochronaut: Thank you for sharing you experience! It looks as if the components cardioid condenser, dark field stop, objective aperture and funnel stop were used for acheiving dark field. For dark field stops I know only of applications up to max. a 40:1 objective. Cardiod condensers have apertures bands from ca. 1 to 1,4 but there were also weaker versions. I know of oil immersion objectives 90:1 0,8-1,25 and 60 0,6-1,0. Which combinations have been suggested by the different makers? Was there a combination of cardioid condenser and funnel stop together? I have to have a closer look: I have an english Baker? objective 80:1 Apo n.a. 1,32 that was intended to be used with a funnel stop iirc. I think the description is enclosed and will post here what they suggest.

My 90:1 0,8 - 1,25 achromat is an old pre war Zeiss design with quite heavy field curvature. With a funnel stop I could use more modern objectives for dark field.

Bob
Not sure what you mean by "was there a combination of cardioid condenser and funnel stop together". Perhaps you mean, were there kits sold, with both in them ?
The answer to that would be yes. Leitz, at one time sold a cardioid condenser along with an iris fitted objective upper section, kit. The iris section would just screw onto the lower lens section of the higher N.A. objectives. Other companies, have usually sold the condenser and a selection of funnel stop shapes to be used with whatever objectives would normally have been used with that condenser, in addition to offering iris equipped objectives as well. Both Spencer and Bausch & Lomb offered 1.25 and 1.30 N.A. objectives with built in restrictor baffles, to bring the N.A. down to .85 and .80 respectively. AO at one time supplied 4 different funnel stops in the condenser case, and they could be used with about 6 different objectives, including a couple made by Bausch & Lomb.
Objectives with a full barrel, where the back lens is very close to the upper end of the barrel, have sometimes had stops made for them that replace the rear baffle plate, where that plate is fitted in as a baffle only( not a lens housing). Bausch & Lomb had such a device for their early flat field achromats and I successfully swapped the rear baffle from a D.I.N. Reichert 100X 1.25 plan objective to a D.I.N. Reichert 63X 1.0 plan glyzerin immersion objective, effectively reducing the N.A. enough, so it could image DF with a crisp dark background. It is hard to make a precise funnel stop but replacing the rear baffle with a restrictor baffle is a little easier.

Funnel stops and restrictor baffles have surprisingly small apertures. The ratio of full aperture to that of restricted aperture would also be affected by the location of the restriction in the light path. Presumably, funnel stops and or restrictor baffles manufactured in the factory, would be optimized for the maximum dark background vs. resolution quotient, because excessive restriction results in a drop in resolution, rather than an increase.

You don't achieve perfect DF by bringing the N.A. down to the lower limit of the condensers, N.A. You have to get below it. There is a border of dimishment, then a diminishing pale grey to black background as the N.A drops, because light creeps in via reflection,flare and aberrations, when the objective aperture and condenser aperture are very close. Usually, and this is somewhat dependent on the sample thickness, cleanliness of the hardware, objective and condenser design, integrity of the oil etc., one needs to drop the N.A. 1-2 points below the lower limit of the condenser, in order to optimize the contrast and resolution.

Here are some measured diameters at funnel stop tip( which sits right at the rear lens of the objective) , the original diameter at the rear lens aperture, the aperture of the rear exit of the funnel stop and the diameter of the original rear baffle aperture of some objectives I have with funnel stops. I also include the diameter ratio between the original rear baffle and the restrictor baffle I use for DF on the 63X 1.0 plan glyzerin immersion objective. Without using the restricter, that objective's image is crowded by low contrast and flare all around it's periphery. With the restrictor , it gives a very dark background and excellent resolution.

B & L 97X 1.30 oil achromat. early 60's manufacture. funnel nose aperture .125( thou.), rear lens diameter .275( +- a bit), funnel exit aperture .193, normal exit aperture .290. length of funnel stop 1.220 35.7mm parfocal objective ( or so I have read, such)

AO/Spencer 97X 1.25 oil achromat. late 50's manufacture. funnel nose aperture .105, rear lens diameter .170(+-a bit), funnel exit aperture .316 , normal exit aperture .238. length of funnel stop 1.228. 34 mm parfocal objective.

AO/Spencer 100X 1.25 oil achromat( cat.# 1079). 60's to 90's manufacture, infinity corrected. funnel nose aperture .103, rear lens diameter .170(+-a bit), funnel exit aperture .316, normal exit aperture .235, length of funnel stop 1.224. 34mm parfocal objective.

Reichert 63X 1.0 oil achromat . 60's manufacture. funnel nose aperture .116, rear lens diameter .222(+- a bit), funnel exit aperture .277, normal exit aperture unknown, length of funnel stop 1.207 37mm parfocal objective.

Reichert 63X 1.0 plan glyzerin. 80's manufacture. restrictor baffle .213, normal baffle .235. D.I.N. infinity corrected objective.

Spencer 95X 1.25 oil achromat w/. iris. 40's manufacture. iris diameter .110(+- a bit) when the iris is closed to a .80 setting, diameter of the rear lens when iris fully open .180, exit aperture .238. 34mm parfocal objective.

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Re: Dark field illumination: Questions and answers

#5 Post by Hobbyst46 » Thu Mar 01, 2018 7:27 pm

wporter - Thanks for the remark about collimated beams. Actually, I was thinking about the non-collimated beam between the lamp and collimating optics. My original light bulb as well as its LED replacement are located near the frosted lens of the collector optics. There is no concave mirror behind the lamp, and I was somehow (theoretically) considering a more powerful LED further away from the frosted lens.
Last edited by Hobbyst46 on Thu Mar 01, 2018 9:56 pm, edited 1 time in total.
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Re: Dark field illumination: Questions and answers

#6 Post by MicroBob » Thu Mar 01, 2018 8:54 pm

Thanks Apochronaut for your detailed information!
I looked at two objectives this evening:

1. Cooke 80:1 1,32 Apo Oil with installed darkfield funnel and description
2. Vickers 50:1 1.0 Fluo Oil

With the Zeiss Jena pancratic condensor with the cardioid dark field head it did some tests.

1. The Cooke 80 gave a nice dark field immediately at 10W, a little dim. This means that the darkfield funnel has to be installed. I was not able to open the back of the objective by hand so I left it for now. I show some pictured of the objective and the description. These english objectives look somewhat strange and hand made like. I have not tested them thoroughly so far. The first impression was good

2. Vickers 50:1 0,95 Oil Flu gave a nice dark field immediately at 10W, brightness correct. I think this objective was aimed at the medical work to look at blood smears. Very good first impression, quite promising. 50 1,0 objectives give a nice crisp view and offer a bigger field of view. They are also nice next to a 100 Oil.

The last picture made me think: Amphipleura pellucida was an object I worked on before and here they are in a sample where I didn't expect them.... :roll: Is this species everywhere or do I have to clean my stuff better?

The sample is from a canal in Hamburg, brackish water, cleaned with heat.

Bob
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Re: Dark field illumination: Questions and answers

#7 Post by MicroBob » Thu Mar 01, 2018 8:56 pm

Last pictures of the Cooke 80
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Re: Dark field illumination: Questions and answers

#8 Post by Hobbyst46 » Thu Mar 01, 2018 9:49 pm

Awsome pitch-dark background! Thanks very much for posting these DF, Bob.

Just a thought: A couple of years ago, phase contrast by means of a LED ring instead of the phase contrast condenser was described; when will they create darkfield by oriented LED light rather than condensers, reflectors and stops ??
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Re: Dark field illumination: Questions and answers

#9 Post by apochronaut » Fri Mar 02, 2018 2:09 am

Nice objectives .
Baker had some sort of relationship with AO , prior to AO's investment in Reichert . A fair amount of Baker and AO hardware from the 50's have a great amount of similarity. I am sure there was some cooperation between the two companies going way back. AO even made a version of the Baker Interference microscope, with Baker optics on an AO stand. They both were 34mm parfocal. AO had an 82X 1.30 apochromat at one time . All of the earlier separable AO objectives that required a funnel stop for DF used a small one that dropped in after the bottom lens section was separated. If you unthread the lens section of your Baker, basically red ring and below, you will find a funnel stop in there. You may have tried this objective for BF in the past and found it very disappointing, as it would be with the N.A. reduced to .80 or .85. You will find a big difference in BF with the funnel stop removed.

Have you looked into the rear of the 50X fluorite to see if there is a funnel stop in there. It would be unusual to obtain such a dark background at .95.

Baker became Vickers, I think in 1963. It had been owned by Vickers for some time, possibly back into the 40's or earlier.. When Cooke Troughton and Simms merged with Cooke or , I think might have been absorbed, the moniker Baker Cooke Troughton and Simms seemed a little much so they just used Vickers. By then, they were already making D.I.N. optics.

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Re: Dark field illumination: Questions and answers

#10 Post by MicroBob » Fri Mar 02, 2018 6:22 pm

@Hobbyst46: I spent some thinking on a darkfield LED-ring, actually made a rough prototype a couple of years ago. I can't see any reason why this shouldn't work. The companies won't make such an condenser because dark field is not so well respected any more because of the artefacts it produces. Institutions and companies that would buy expensive microscopes new will look for contrast techniques that induce as little artefacts as possible (and need the least amount of exerience...). One area where dark field microscopy is hyped very much is live blood microscopy. There was an especially interestig post on this forum about this controversely discussed topic.

@Apochronaut: I will have a look for a funnel stop in the 50:1 0,95 Vickers. I also have a Leitz NPL Fluotar 50:1 1,0 Oil and can compare these two.
These english makers had these microplan, fluo and apo objectives that are unusual for a low volume maker. So it would be a likely explanation that they had a bigger company as a partner here. I don't kow how much calculation was needed in 1950 or 1970 to design these objectives. A simple achromat was only two pages of calculations by hand or so. These advanced objectives might have been unobtainable without a cooperation. It is interesting anyway how many microscope makers of considerable quality there were until the 70s. Nearly every country had an own microscope company. Maybe this was seen as a status symbol or as important for military or business affairs. I suspect that there was more money spent than a self sustaining company would have seen as acceptable.

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Re: Dark field illumination: Questions and answers

#11 Post by apochronaut » Sat Mar 03, 2018 12:21 am

With Vickers , they had considerable operations otherwise. Microscopy ended up in their lap as an acquisition and would have been of interest to them principally for metallurgy and measuring. Their biological unit sales dwindled over time.

Cooke always had an optical base and still does. They are one of the best in the world. I'm not sure where or when the Cooke camera lens and Cooke microscope lens concerns cleaved off but the ability of a company with Cooke in the name to make optics as good as anyone, isn't surprising.

I have a few of those older Cooke Baker and Vickers objectives around., a 100X 1.30 DF water immersion( seems to have a built in aperture restrictor), a 50X 1.0 water immersion , some Vickers Microplan achromats. They are all good.

Many other companies were arms of a larger company's more vibrant operations. Bausch & Lomb is one of the biggest companies in the world today, based almost entirely on contact lenses. They didn't really miss the scientific apparatus division, when Cambridge bought it.

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Re: Dark field illumination: Questions and answers

#12 Post by MichaelG. » Sat Mar 03, 2018 9:38 am

apochronaut wrote:Cooke always had an optical base and still does. They are one of the best in the world. I'm not sure where or when the Cooke camera lens and Cooke microscope lens concerns cleaved off but the ability of a company with Cooke in the name to make optics as good as anyone, isn't surprising.
Useful 'timeline' here:
https://www.cookeoptics.com/t/history.html

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Re: Dark field illumination: Questions and answers

#13 Post by einman » Sat Mar 03, 2018 2:37 pm

Apo in reference to a Leitz component:"The iris section would just screw onto the lower lens section of the higher N.A. objectives."

I actually have one of these although I have never used it.

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Re: Dark field illumination: Questions and answers

#14 Post by charlie g » Sat Mar 03, 2018 8:39 pm

I hope this is a :'dark field Q&A thread'...sorry if I misunderstood OP intent.

I have a wonderful vintage (? pre WWII?) Emst Leitz Wetzlar dedicated dark field condenser. This component has no iris mechanism...my substage field illuminator has both an iris control lever/ field iris, and a focus control simple field lens group with adjustment lever..so this controllable field illuminator component ( Reichert/Austria)...functions well with the dedicated vintage DF condenser.

My questions: 1) when a dedicated vintage df condenser has no iris...does this imply it is mounted with a substage iris snug below it? Or also imply a field illuminator has the focus control lens group+an adjustable iris to 'feed illumination' into this dedicated vintage df condenser ( NA: 1.20 , the etched spec.)?

2) The lens face of this vintage df condenser ( which is right adjacent to a stage slide lower surface) is broad and flat...is this a condenser surface which needs oil-bridge to slide ..by the spec. being: N.A. 1.20?

3) Is this df condenser dependant on a specific tube-length microscope? Or are substage condensers independent of a stands: tube length spec? As long as this df condenser is setup properly with oil-bridge to a slide..the focus control field illuminator+ field adjustable iris are the only important optical criteria...and not the 'tube length spec of the microscope above this oil-bridged slide setup?

4) In general sense..whenever a substge condenser ( df or multiple method condenser) is oil-bridged to a slides undersurface...is it pointless to require a 'tube-length spec' for these variety of finite optical substage condensers?

Thanks for any comments or thoughts ( I hope it appropriate that I nested my images and concerns in this excellent and useful thread). Charlie guevara
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Re: Dark field illumination: Questions and answers

#15 Post by charlie g » Sat Mar 03, 2018 9:09 pm

The 'conic sections of this df condensers illumination output...are quite adjustable with the field-illuminator having an iris+ adjustable focus of it's tungsten flat filament ( Oshram flat-filament tungsten bulb).

Providing proper substage mount, proper substage 'rack&pinnon track length permint'...are these vintage oil-bridge dedicated condensers...and other finite optical condensers independent of a stands specific tube-length spec? Charlie guevara
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Hobbyst46
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Re: Dark field illumination: Questions and answers

#16 Post by Hobbyst46 » Sat Mar 03, 2018 9:27 pm

Charlie g: I also believe that this thread is DF Q&A. And somehow all solutions are based on vintage, rare sophisticated condensers and objectives...
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Re: Dark field illumination: Questions and answers

#17 Post by MichaelG. » Sat Mar 03, 2018 9:50 pm

charlie g wrote:My questions:

3) Is this df condenser dependant on a specific tube-length microscope? Or are substage condensers independent of a stands: tube length spec? ...
charlie g
I can see no optical reason why the condenser should be specific to a certain tube length.
... I'm always ready to learn, though !

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Re: Dark field illumination: Questions and answers

#18 Post by MicroBob » Sun Mar 04, 2018 8:53 am

Hi Charlie,

thank for showing this nice piece of equipment! Your are perfectly right here in this thread. My idea was to discuss general darkfield topics here as they are often related.
To your questions:
1) A normal dark field condenser has no iris at all. It offers a hollow light cone of e.g. n.a. 0,9 to n.a. 1,2 and is used like that. If there is an iris it remains to be seen what it does.
2) All condensers of n.a. 1 and over need to be immergated to the slide. There is a total reflection of the flat angled rays otherwise.
3) +4) The condenser is independent of the tube lenth to the eyepiece. It lights the slide in a certain way. What happens next is independent. This is general for all condensers I can think of.

Where condensers are different is the lightpath from lamp to condenser. Some condensers are designed to pick up a lighted aperture 25cm behind the mirror or so. If you replace mirror and external lamp with a flat LED lamp directly on the foot of the stand the condenser is used outside of his specification and doesn't reach the specified aperture. This is the case with the Zeiss Standard Junior microscopes. If you use a PZO or Leitz condenser instead you get a proper lighting.

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Re: Dark field illumination: Questions and answers

#19 Post by MicroBob » Sun Mar 04, 2018 8:57 am

Dark field illumination and micro photography: Exposure meters are set up for 18% medium gray objects. In a dark field image there is mainly black, so the exposure will be wrong. By adjusting the exposure so that the object itself has the right brightness you also get a darker background. Most digital cameras have a +/- setting where you have to dial in about 1-3 stops less light.

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Re: Dark field illumination: Questions and answers

#20 Post by Hobbyst46 » Sun Mar 04, 2018 10:47 am

when will they create darkfield by oriented LED light rather than condensers, reflectors and stops ??
That was a dumb question! Just found out that such devices are not new and are being sold,

https://www.edmundoptics.com/illuminati ... luminator/

Although not inexpensive, and perhaps not for any microscope stand...

Another ref:
Jorg Haus : Darkfield Microscopy with HighPower LED Illumination, Optical design benefits from replacement of halogen lamps
Optik & Photonik Volume 10, Issue 1, Version of Record online: 16 FEB 2015
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Re: Dark field illumination: Questions and answers

#21 Post by MicroBob » Sun Mar 04, 2018 2:27 pm

@Apochronaut: I had a look at the Vickers 50 1.0 oil and couldn't see a funnel. I then compared to the Leitz NPL fluotar 50 Oel 1.0 and again got a nice dark field image. So I think that the Zeiss Jena pancratic condensor produces a hollow light cone with n.a of clearly over 1,0 to 1,4. I have no real experience with this impressive piece of optical kit. I have two of them, one is stuck at full aperture, one at minimum aperture. :lol: I really have get them going since the results look so promising.

@Hobbsyt46: This Edmund optics part would not be difficult to copy. It is only for low power darkfield work, offers probably less then a normal abbe condenser with dark field stop. More interesting would be a condensor made from an acryl glass body with LEDs shining into it from the side. I had a look for my prototype but couldn't find it. It would be quite a bit of work and precise machining to make one of these in a form ready for everyday use. So it's not that interesting if there is a used market that supplies dark field condensers for acceptable money.

Enlosed two nice darkfield images with the Leitz NPL Fluotar 50 1.0 Oel
All single shots, camera set to -3 stops, no stacking, only cut out (severely), resized, sharpened, no editing
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Re: Dark field illumination: Questions and answers

#22 Post by Hobbyst46 » Sun Mar 04, 2018 3:11 pm

Very nice photos.
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apochronaut
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Re: Dark field illumination: Questions and answers

#23 Post by apochronaut » Sun Mar 04, 2018 3:17 pm

charlie g wrote:I hope this is a :'dark field Q&A thread'...sorry if I misunderstood OP intent.

I have a wonderful vintage (? pre WWII?) Emst Leitz Wetzlar dedicated dark field condenser. This component has no iris mechanism...my substage field illuminator has both an iris control lever/ field iris, and a focus control simple field lens group with adjustment lever..so this controllable field illuminator component ( Reichert/Austria)...functions well with the dedicated vintage DF condenser.

My questions: 1) when a dedicated vintage df condenser has no iris...does this imply it is mounted with a substage iris snug below it? Or also imply a field illuminator has the focus control lens group+an adjustable iris to 'feed illumination' into this dedicated vintage df condenser ( NA: 1.20 , the etched spec.)?

2) The lens face of this vintage df condenser ( which is right adjacent to a stage slide lower surface) is broad and flat...is this a condenser surface which needs oil-bridge to slide ..by the spec. being: N.A. 1.20?

3) Is this df condenser dependant on a specific tube-length microscope? Or are substage condensers independent of a stands: tube length spec? As long as this df condenser is setup properly with oil-bridge to a slide..the focus control field illuminator+ field adjustable iris are the only important optical criteria...and not the 'tube length spec of the microscope above this oil-bridged slide setup?

4) In general sense..whenever a substge condenser ( df or multiple method condenser) is oil-bridged to a slides undersurface...is it pointless to require a 'tube-length spec' for these variety of finite optical substage condensers?

Thanks for any comments or thoughts ( I hope it appropriate that I nested my images and concerns in this excellent and useful thread). Charlie guevara
That is the Leitz kit, I was referring to. I too have one, which seems unused. DF seems to have been one of those techniques that is a bit daunting to some. Condensers or condenser kits seem to have been purchased and only minimally or never used.

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Re: Dark field illumination: Questions and answers

#24 Post by einman » Sun Mar 04, 2018 3:43 pm

Very nice photos!

I recently acquired a Leitz NPL Fluotar 63x/ 0.6-1.3 oil objective with an iris. Somewhat rare I understand. I was looking for the 50X/1.00 oil and stumbled across this one instead. It came with a 25X/0.75 phaco 2 Fluotar oil immersion objective which I put on E-bay. Not certain I wish to sell it so I priced it high, or so I thought. There is another one listed for more than twice as much!

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Re: Dark field illumination: Questions and answers

#25 Post by charlie g » Sun Mar 04, 2018 4:02 pm

That second diatom image capture is stunning...so crisp a sense of that frustule, Microbob...thank you! Charlie guevara
Last edited by charlie g on Thu Mar 08, 2018 4:08 am, edited 1 time in total.

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Re: Dark field illumination: Questions and answers

#26 Post by charlie g » Sun Mar 04, 2018 4:59 pm

So are objectives which have an adjustable iris for use with other contrast methods, as well as dark field contrast method? Is a simple function of an objective with internal iris..simply to enhance contrast..but with limitations on resolution as the iris is closed down...as the objectives I thought higher NA objectives had an adjustable iris to act as a muti-setting: 'funnel stop'? I thought an objective with an inserted ( and thus removable) funnel stop..was equivalent to a higher NA objective lacking a correction collar...it is a setting of the optics to cover 'most' of the conditions in an objective in optical use...but a compromise to keep costs down...the correction-collar high dry objective...or the objective with an iris are both costly add ons to maximize the optical performance of a high NA objective.

I am trying to ask: 1) are not set/specific inserted funnel stops a compromise...just as high NA dry objectives lacking a correction-collar..are compromizes? I thought an objective tasked with high NA dark field contrast method..will best be setup for maximum optical resolution, contrast...by use of an adjustable iris in the high NA objective?

2) Is it being stated that the best NA a dedicated DF condenser functions with is: NA 1.2 ?

So what function does my 90X / NA 1.25 with adjustable iris have as it's desired optical setup? I in simple error always thought: 'adjustable iris high NA objectives...are for optimum use as dark field contrast method thanks, Charlie guevara
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wporter
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Re: Dark field illumination: Questions and answers

#27 Post by wporter » Sun Mar 04, 2018 5:47 pm

As far as I know, iris-equipped high-power objectives are only designed for darkfield work: the iris enables one to lower the NA of the objective to under that of the darkfield condenser, so that no direct light is captured by the objective. Of course, this doesn't preclude you playing around with the objective iris for fun! As you close down the objective iris, the background might turn from grey to black as your objective NA approaches, then goes below, your condenser NA.

(The term 'correction collar' should only be used for those objectives that have movable elements inside the barrel that are shifted by the operator to compensate for different coverglass, or (in the case of inverted microscopes) different culture-well, bottom thicknesses. It is kind of confusing, since both type of objectives have knurled collars, but generally the true 'correction collar' will have a graduated scale indicating different coverglass thickness settings.)

Further, special high-NA darkfield condensers must be oiled to the bottom of the slide; otherwise, no light at all will escape the condenser to illuminate the specimen.

There is a great writeup on setting up darkfield here:
https://www.med.unc.edu/microscopy/reso ... -formation

(Select Ch-9, Dark Field for the pdf document))

apochronaut
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Re: Dark field illumination: Questions and answers

#28 Post by apochronaut » Sun Mar 04, 2018 6:18 pm

Charlie g : my take on your questions. 1) It seems in DF , there is an optimum aperture where the contrast and resolution are both nicely balanced. I would presume that most funnel stops or restrictor baffles( fixed diaphragms) are designed to be at that optimization. I have spent some time adjusting iris diaphragms to optimize DF with about 6 different objectives. The results were similar with each objective. Contrast increased to a maximum , as the iris was closed. While it would be hard to argue that the resolution increased as the iris was closed as well, the apparent resolution did. Initially with an open iris, low contrast and flare, mask the resolving power of the objective. As the iris is closed, there seems to be an increase in resolution down to a point just above the point of maximum contrast, where it falls off rapidly. Based on these observations, I see the apertures of factory made funnel stops to be optimized for most conditions with a recommended condenser, rather than a compromise.
There is another feature of a closed iris or funnel stop. It increases the depth of field. There have been times I have seen, with samples of very small subjects in DF, that close to the entire depth of an aqueous sample is in relatively good focus.
Iris diaphragms have in the past, been used for just such a purpose, increasing the depth of field for photomicrography use, similar to closing the aperture in a camera lens. One objective I have acquired over the years, is an almost 100 year old, Spencer 8mm 20X .50 with an iris graduated from .2 to .5 N.A. While this is not necessary for DF, it is useful to increase depth of field. There is virtually no perceptible reduction in resolution as the iris is closed, until you get just below .4. It is easy to see that, at that level of reduction in the N.A., it could be useful for certain photography applications. In more modern microscopy, iris diaphragms are very useful in fluorescence, . An iris does give more control over the various factors it affects but funnel or diaphragm stops, seem pretty optimized for DF.
2) No. Condensers vary and the best N.A. to optimize DF with any given condenser will vary but most cardioids are quite similar and most parabaloids, etc. I have read literature time and time again, that indicates that an objective need only have an N.A. below the lower limit of the condenser to achieve DF but in practice, I have not found that gives the best DF, unless everything in the system is at a complete optimum; slide thickness, cover glass thickness, cleanliness, sample thickness, sample n, and on and on. Most of the oil immersion high resolution DF I have done has been best at up to .2 below the lower limit of the condenser , sometimes less. Typically, .8 to 1.00 gives the best results. One can go higher sometimes but the loss of contrast related to opening up the aperture with wide fields is counterintuitive. Flare enters from the perimeter, so there is some advantage in DF, to having a narrower F.N.. To use your N.A. 1.25 objective for DF, you will need to use the iris.
Last edited by apochronaut on Mon Mar 05, 2018 12:52 pm, edited 1 time in total.

MicroBob
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Re: Dark field illumination: Questions and answers

#29 Post by MicroBob » Sun Mar 04, 2018 6:36 pm

@wporter: Thank you for the link, a really good description!
The author states that dark field became popular after someone identified the syspillis bacterium in 1905. I think dark field has been superceeded by phase contrast after WW2, so fewer newer dark field condensers on the market.

@Apochronaut: The old times film photomicrography is an interesting new aspect. When stacking was not available it is easy to see that the microscopist was happy about one more option to adjust his picture. Overall objectives with an iris are quite rare. I personally have never recognized anything but high power oil immersion objectives in ebay auctions, but I am not looking closer at really old objectives since I'm not a collector. Maybe the additional cost of the iris was too high for what the market would allow.

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Re: Dark field illumination: Questions and answers

#30 Post by wporter » Sun Mar 04, 2018 7:39 pm

Apo is completely right, my mistake; iris objectives are also useful in fluorescence work. My Univar manual says, in fact, for both transmitted-light brightfield and darkfield fluorescence: "With strongly fluorescent preparations, the objective iris can be closed down to control glare."

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