Phase Microscopy part II

Here you can discuss different microscopic techniques and illumination methods, such as Brightfield, Darkfield, Phase Contrast, DIC, Oblique illumination, etc.
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apochronaut
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Phase Microscopy part II

#1 Post by apochronaut » Thu Mar 12, 2020 10:47 am

In the previous entry about phase contrast, I covered a bit of the history and general principals. It was noted that phase contrast is not one technique but highly variable : really it can be as many as are imagined. Sadly, it has become one thing, with most companies opting to produce a general dark phase system.
In part II, I am covering the general design parameters that when changed or adjusted , alter the type of phase and or the contrast level. Different phase types yield differing results with various types of samples, even different thicknesses of the same sample, so it is clearly evident that the dearth of phase contrast types on the market today, is a detriment to quality microscopy, since phase is one of the least expensive variable contrast tools available. Even different levels of contrast have applications separate from one another. Thus phase contrast is a continuum. For brevity, I will be describing a theoretical system that has an annular design, although phase systems with designs other than annular have existed. Thus, the functional parts of the system are called an annulus or annuli.

A phase system begins with a light source that has conjugate planes at the condenser diaphragm( the phase annulus in the phase condenser body), the focus plane( the object plane) and the diffraction plate( the phase annulus in the objective or above the objective, whichever is the manufacturers method). This is called the conjugate plane or the conjugate area.

Additionally, the diaphragm and diffraction plate are fitted with a complementary area. This is the area of the diaphragm outside of the annulus and the area of the diffraction plate outside of the annulus.

The conditions of the diapragm can be changed to yield different results and the conditions of the diffraction plate can be changed to yield different results.

The diaphragm can be adjusted as to the width and size of the annulus. The width and size of the diaphragm annulus can be adjusted relative to the width and size of the diffraction plate annulus to yield different results or the diffraction plate annulus and diaphragm annulus ratio can remain constant but of different proportions and that will yield different results as well.
The objective diffraction plate can be adjusted as to the degree of absorption in both the conjugate and complementary areas and the degree of wave separation in both the conjugate and complementary areas. Adjusting these parameters, will also yield different results.

Given the high degree of variability available to the design of a phase system, it seems surprising that the industry has defaulted to such a small selection of phase options. It is an area very much open to diy engineering.


Below are 4 representative diffraction plates in cross section, showing the absorptive and diffractive layers. Each displays 3 sections of the conjugate area, which represent the central circle and the two outer sections of the outer ring. The two sections of the annular ring are shown as the two smaller sections. The relative size of the various areas on the plate could of course be of any dimensions, as long as the diaphragm was designed in a complementary fashion.

In the images below the conjugate area is designated 0 and the complementary area 1. Images are from Phase Microscopy by Bennet, Jupnik, Osterburg and Richards.
Attachments
Plate with an untreated complementary area and a conjugate area with both absorptive and diffractive properties.
Plate with an untreated complementary area and a conjugate area with both absorptive and diffractive properties.
DSC03132 (1280x544).jpg (123.68 KiB) Viewed 5699 times
Plate with a diffractive complementary area and a conjugate area with absorptive properties.
Plate with a diffractive complementary area and a conjugate area with absorptive properties.
DSC03133 (1280x546).jpg (112.54 KiB) Viewed 5699 times
Plate with both a diffractive and absorptive complementary area and an untreated conjugate area
Plate with both a diffractive and absorptive complementary area and an untreated conjugate area
DSC03134 (1280x556).jpg (120.73 KiB) Viewed 5699 times
Plate with an absorptive complementary area and a diffractive conjugate area.
Plate with an absorptive complementary area and a diffractive conjugate area.
DSC03135 (1280x545).jpg (112.71 KiB) Viewed 5699 times

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daruosha
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Re: Phase Microscopy part II

#2 Post by daruosha » Thu Mar 12, 2020 2:09 pm

apochronaut,

Awesome series. It's very useful to know the different versions of Phase Contrast and how they are constructed. Unfortunately manufacturers, even the big ones usually don't mention the type of their phase contrast objectives and you don't know what are you buying exactly. I looked through Olympus catalog and couldn't find any details about how their objectives render the final contract (e.g. dark, positive, ...). They are PL (I assume it's positive low).

For the sake of compliments, I link your previous topic of your Phase Contrast series:
viewtopic.php?f=28&t=8752
Daruosh.

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daruosha
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Re: Phase Microscopy part II

#3 Post by daruosha » Thu Mar 12, 2020 2:16 pm

There is also a very well written article by MicroscopyU.com website I would like to add:

https://www.microscopyu.com/techniques/ ... microscopy

apochronaut, I hope it's not an intervention in your posts. Apologies in advance :).
Daruosh.

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Re: Phase Microscopy part II

#4 Post by BramHuntingNematodes » Thu Mar 12, 2020 2:38 pm

These are excellent posts and I hope you will continue them. I would someday like to experiment with some phase contrast using my microscope lathe, so historical examples of different methods and materials used is extremely heartening and helpful.

I have a single phase objective, it is a Wild achromat with the spring-loaded front lens assembly, and works well with my old B&L compens eyepieces. One thing I have thought is that the aspiring phase contrast engineer can detect materials that induce the appropriate phase changes with the help of...a microscope set up for phase contrast!
1942 Bausch and Lomb Series T Dynoptic, Custom Illumination

apochronaut
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Re: Phase Microscopy part II

#5 Post by apochronaut » Fri Mar 13, 2020 9:35 am

Thanks to all. I will probably post one more. I'm still experimenting with various phase types and moderating things a bit by making some odd phase annuli adjustments plus trying out objectives designed for other systems. Using Reichert Austria infinity phase objectives with a Reichert U.S.A. infinity, condenser diaphragm set has provided a few hours of education., The differences in the diffraction plate construction and location in the objective seem to be the most influential part of the system.
The AO type B-Minus system is noteworthy in that you cannot visually see the annular ring on the diffraction plate. Alignment is tricky. The best way I have found to do it is to center the objective set on a separate nosepiece, which is physically centered to match some other type of phase objective set that does have visible annular rings. In my case I have Dark medium and B-Minus medium matched on separate quick change nosepieces. As long as the objectives themselves are manufactured so as to be precisely centered and one would presume that they are; once the Dark objectives have been aligned with the bertrand lens, swapping in the B -Minus should maintain the alignment.
The recommendation is to adjust the system based on contrast level but I haven't found that to be necessary using the nosepiece matching method.

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Re: Phase Microscopy part II

#6 Post by Hobbyst46 » Fri Mar 13, 2020 12:49 pm

apochronaut wrote:
Thu Mar 12, 2020 10:47 am
...It was noted that phase contrast is not one technique but highly variable : really it can be as many as are imagined. Sadly, it has become one thing, with most companies opting to produce a general dark phase system...
Different phase types yield differing results with various types of samples, even different thicknesses of the same sample, so it is clearly evident that the dearth of phase contrast types on the market today, is a detriment to quality microscopy, since phase is one of the least expensive variable contrast tools available.
Thus phase contrast is a continuum.
Given the high degree of variability available to the design of a phase system, it seems surprising that the industry has defaulted to such a small selection of phase options. It is an area very much open to diy engineering.
Thanks for an enjoyable introduction to practical phase contrast. I Hope that you show us more descriptions and examples of the various PC modes.

Indeed, continuum is the key. It offers possibilities. Research microscopy involves a lot of image analysis, especially automated batch quantitative analysis of large image stacks. Identification and selection of a certain type of cell in a tissue, for example. Phase contrast images can sometimes do better than DIC in such instances.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

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Rossf
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Re: Phase Microscopy part II

#7 Post by Rossf » Sat Mar 14, 2020 3:11 am

Very interesting Apochronaut...if you want to and have time do you want to post some images of what some of the unusual A.O phase types look like imagining specimens? I personally think phase still has plenty of life in it post DIC and I suspect a clever company may work on phase 2.0 so to speak. Maybe different plate designs and materials-Olympus tried with the relief phase-but never saw photos that made me go “wow that’s an improvement”,likely why it didn’t last long-maybe I never saw the right photos...did A.O make those negative phase objectives that have a dark circle plate instead of a donut shaped plate?
Regards ross

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Re: Phase Microscopy part II

#8 Post by apochronaut » Thu Mar 19, 2020 12:52 am

Here are some pretty unpolished images but you can get the general idea. The subject is some species of Fragillaria, likely.

These were under pretty high magnification . AO were the only mfg. of B -Minus phase and only made B -Minus in the infinity series of objectives as a 100X 1.25 achromat. I made a comparison between Dark phase, Bright phase and B-Minus phase at 100X 1.25 . Only the dark is a planachro. They are all likely medium contrast but that wasn't published.

I have some phase variants out of the series 400 system but without going to a wholesale head and photo tube conversion , I couldn't image those corrected properly in the older infinity system. I will do those at a later date in a Diastar.

The images below are pretty heavily cropped and probably represent about a 2000X magnification , if viewed on an average laptop.
Attachments
Dark phase planachro . 100X 1.25 oil immersion. The space between Each cell is about 5 microns.  Individual  details are down to .2 microns or so.
Dark phase planachro . 100X 1.25 oil immersion. The space between Each cell is about 5 microns. Individual details are down to .2 microns or so.
DSC03151 (1280x764).jpg (166.32 KiB) Viewed 4795 times
Bright phase achromat. 100X 1.25  oil immersion. resolution details as above.
Bright phase achromat. 100X 1.25 oil immersion. resolution details as above.
DSC03154 (1280x871).jpg (161.71 KiB) Viewed 4795 times
B-Minus phase achromat. 100X 1.25 oil immersion. resolution details as a bove.
B-Minus phase achromat. 100X 1.25 oil immersion. resolution details as a bove.
DSC03153 (1280x788).jpg (143.63 KiB) Viewed 4795 times

Gimble88
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Re: Phase Microscopy part II

#9 Post by Gimble88 » Fri Apr 24, 2020 1:04 am

I hope a question or two is appropriate here. I use Nikon S series microscopes and just acquired a beater with a 77040 (catalog #) phase contrast condenser, 1.3 N.A. The beater came with a vanilla 10/0.3 DLL and plan 40/0.65 DLL. Both are like other 60's S series objectives and a bit modest in performance . So I would like to replace these S series 10x and 40x with maybe CFN plan achros and get a 20x. I have upgraded my bright field microscope to CF(N) objectives and noticed a huge improvement. Used fluor objectives from this series seem to be a bargain. Not much more than plan achros. My question (FINALLY) is: will the newer CF(N) objectives still be compatible with my old S series p/c condenser? I have noticed that a plan 10x/0.3 of the CFN series is marked ph1 while a Fluor 10/0.5 is marked ph2. This would seem logical to account for the much higher NA. But... will it work? I probably wouldn't spring for the fluor objective for phase contrast but I do have a fluor 10/0.5 bright field and to my eye it's wonderful. I'm not too bothered by the smallish field curvature and... we have stacking.

One more question. The condenser annulus marked on the condenser as "40" ( I guess that would be ph3) is not (no longer?) an annulus. The center dark spot is gone leaving just a big hole. No phase contrast effect for the 40x. Is this correct that is once was a ring not a hole? Any ideas about fixing this?

Thanks for your patience,

spence

Gimble88
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Re: Phase Microscopy part II

#10 Post by Gimble88 » Fri Apr 24, 2020 1:52 am

I have to laugh. I unscrewed the condenser lens from the phase contrast wheel and found the little center of the 40 annulus just sitting next to the annulus mounting. At least I know what size it is. It's just paint that flaked off and it's too fragile to handle. A piece broke off when I tried to pick it up with tweezers, but not before I got my dial caliper on it. Luckily the full diameter is still available to measure, 6mm.
spence

apochronaut
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Re: Phase Microscopy part II

#11 Post by apochronaut » Fri Apr 24, 2020 2:29 am

Unfortunately, mixing and matching phase diaphragms and annuli from different systems involves some trial and error, or hopefully finding someone who has travelled that road. I don't know Nikon well enough to be of much help. One way of possibly determining the cross compatibility between the S series condenser and the CF objectives is by part #'s for the respective diaphragms in the two condensers, if you can find them. Another would be by physical measurement, if you can get ahold of the condenser used for the D.I.N. system. That might not tell you anything if they are different anyway , since a different condenser and objective length could compensate for a variance.
Intuitively, because of the objective length difference , I am thinking that they would not be perfectly compatible. However, the resulting imaging could very well be useful.

Well at least you have something to work with. Some of the old ones were painted on glass. I have repaired some with a permanent marker. Completely blank ones are hard to center.

Gimble88
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Re: Phase Microscopy part II

#12 Post by Gimble88 » Fri Apr 24, 2020 3:46 am

Thanks for the quick response. Do you or anyone here have an idea what microscope model would have first used CFN objectives?

spnc

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