"Semi" Dark field?

Here you can discuss different microscopic techniques and illumination methods, such as Brightfield, Darkfield, Phase Contrast, DIC, Oblique illumination, etc.
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smollerthings
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"Semi" Dark field?

#1 Post by smollerthings » Sun Sep 19, 2021 12:22 pm

I really like the colors of dark field but it lacks the clarify and definition and DOF of a stopped down bright field. So I was toying with the idea of getting some kind of semi dark field with a hole in the middle for definition and clarity + outer ring of light to add light and colors.

A bright field + if you will.

It worked pretty well with good clarity + nice colors (you can cut the outer ring by stopping down with the condenser' diaphragm, to really get a sense of what the outer ring of light is bringing to the image)

Is there is a name for that technique? Have you tried it?



Edit: thinking about it, it is probably only useful when the light source is not powerful enough to provide a colorful image at lower condenser stops, like my case.

MicroBob
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Re: "Semi" Dark field?

#2 Post by MicroBob » Sun Sep 19, 2021 5:05 pm

Usually a microscope image is evaluated for contrast and resolution. The small stop in the center should give a lot of conrast combined with little resolution. A real dark field image is quite contrasty and has a lot of resolution too. Can you show images of what you see with the different techniques?

smollerthings
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Re: "Semi" Dark field?

#3 Post by smollerthings » Sun Sep 19, 2021 5:24 pm

I think it is a bit too subtle for me to capture it with my phone and all its automatic settings.

But generally, I do have a resolution issue. For ex below 40x. It is really hazy and I can't close down the condenser diaphragm since it is Rheinberg.
Condenser in top position, fully open


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Re: "Semi" Dark field?

#4 Post by MicroBob » Sun Sep 19, 2021 8:48 pm

The high resolving power of the stronger microscope objectives is accompanied by a verry narrow depth of field. As soon as you try to get a better depth of field by stopping down the resolution will decrease, barely visible at first. The same is true for stopping down to increase contrast. It is better to concentrate on the plane of sharpness and assemble the details in the head (the microscopists's). Fot photograpy stacking is an option but not from fast moving subjects like a rotifier. To get a sharper picture of the cilia it will be necessary to use flash and let it burn for a short time only. Enclosed an image which was the result of a quick flash setup test by me, not at all speciallized in this subject. Contrast will have been DIC (can't remember for sure) but oblique would bring you close. There is a fair amount of detail and contrast, but limited depth of field. If you want to see what the speciallists acheive: https://www.mikroskopie-forum.de/index. ... ic=40943.0
Don't worry if you can't get these results, I can't too, one has to specialize on this subject, gain experience, buy rare and expensive equipment and spend lots of time.
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smollerthings
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Re: "Semi" Dark field?

#5 Post by smollerthings » Mon Sep 20, 2021 7:22 am

Thanks Microbob, very interesting and those pics are very impressive. I think what bothers me is not the shallow DOF but the fact that in some circumstances, what is not in focus gets totally blown out... and this is very distracting.

DevonianSea
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Re: "Semi" Dark field?

#6 Post by DevonianSea » Fri Nov 26, 2021 8:59 am

smollerthings wrote:
Sun Sep 19, 2021 12:22 pm
Is there is a name for that technique? Have you tried it?
Yes there's a name! This seems to be a colour-filtered variant of VBDC (variable brightfield-darkfield contrast), more specifically "condender-based concentric VBDC". This technique was, as far as I know, first named and published in 2011 by German researcher Timm Piper. Refer to Timm Piper's website for more information: http://www.timm-piper.com/Principles_of ... _vbdc.html

He calculated that the area of illumination of the peripheral darkfield component of the light stop should be 89–178x larger than the brightfield component (the central hole, in your case) to get a well-balanced image.

apochronaut
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Re: "Semi" Dark field?

#7 Post by apochronaut » Fri Nov 26, 2021 2:47 pm

It doesn't make a lot of sense to be discussing maximizing DF results while at the same time using stops and a conventional refracting condenser : maybe even an abbe no less. It's kind of like entering an Indy 500 race driving a Fiat 500.
The only way to achieve excellent DF is by using a dedicated reflecting DF oil immersion condenser. Once one has that in hand as well as high resolution objectives wth iris diaphragms or funnel stops, your horizon broadens and all sorts of possibilities become clear. You cannot make a silk purse out of a sow's ear.
With respect to depth of field, utilizing high N.A objectives stopped down to achieve DF, allows for an increase in depth of field in DF, relative to that achieved in BF or Phase for example. This is not the case when using lower N.A. objectives, since they usually can be used at full aperture.
Being able to view almost the entire depth of field of a thin pond smear in high resolution at 1000X in aberration free DF is quite the experience, unlike anything else I have experienced in almost 60 years of microscopy. It does take 4-500.00 to get there, maybe as low as 300.00 , if one aims for older non plan apochromat objectives. DF above all else is heavily distorted by optical aberrations. The designers if DF systems recognized this fact 100 years ago and quickly came up with aberration free optics in order to free DF from the purgatory of distortion. A DF image made with dedicated 1925 DF optics will be far superior than one made with a brand new achromat objective and stopped abbe condenser of today.

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Re: "Semi" Dark field?

#8 Post by josmann » Mon Dec 06, 2021 2:27 am

apochronaut wrote:
Fri Nov 26, 2021 2:47 pm
Being able to view almost the entire depth of field of a thin pond smear in high resolution at 1000X in aberration free DF is quite the experience, unlike anything else I have experienced in almost 60 years of microscopy.
Uh yeah that's a pretty good sales pitch. I think I'll go for this soon. Any advice in particular about prepping the sample/configuring the scope? One thing I'm still not 100% clear on is how to tell when the DF condenser is properly aligned - do I just defocus it a bunch?
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Re: "Semi" Dark field?

#9 Post by zzffnn » Mon Dec 06, 2021 5:48 am

josmann wrote:
Mon Dec 06, 2021 2:27 am
Any advice in particular about prepping the sample/configuring the scope? One thing I'm still not 100% clear on is how to tell when the DF condenser is properly aligned - do I just defocus it a bunch?
Make a very thin mount. Avoid debris or dust.

Oil the condenser and make sure it is FULLY immersed in oil, if it is for immersion darkfield.

Use 100x NA > = 1.25 objective without iris or stopping down to obtain a perfectly concentric COL at objective back focal plane (by removing eyepiece and looking down eyetube). Then go back to lower NA objective, you will have perfectly aligned darkfield condenser.

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75RR
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Re: "Semi" Dark field?

#10 Post by 75RR » Mon Dec 06, 2021 6:17 am

zzffnn wrote:
Mon Dec 06, 2021 5:48 am
josmann wrote:
Mon Dec 06, 2021 2:27 am
Any advice in particular about prepping the sample/configuring the scope? One thing I'm still not 100% clear on is how to tell when the DF condenser is properly aligned - do I just defocus it a bunch?
Make a very thin mount. Avoid debris or dust.

Oil the condenser and make sure it is FULLY immersed in oil, if it is for immersion darkfield.

Use 100x NA > = 1.25 objective without iris or stopping down to obtain a perfectly concentric COL at objective back focal plane (by removing eyepiece and looking down eyetube). Then go back to lower NA objective, you will have perfectly aligned darkfield condenser.
+1

Helps to have a phase telescope or installed Bertrand lens - also, you may want to explore high magnification COL once you achieve it :)
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josmann
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Re: "Semi" Dark field?

#11 Post by josmann » Tue Dec 07, 2021 9:28 am

Looking at the objective BFP definitely helped with alignment. I ordered a CT too since it's such a tiny image.

I tried today with a slide which probably wasn't thin enough - looked like a fair amount of spherical aberration. I know with certainty I can get good results with this lens generally speaking. One issue which was present, though, was an awful lot of diffractive starbursts caused by the objective's iris. I don't expect that would go away fully even with a well prepared slide.

I have read some of apochronaut's previous writings on darkfield and suspect the absence of a gentler field stop is hurting me here. One thing I didn't try since I had already oiled the slide was my 40x .95. I'll do that next time.
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