A brief review of phase contrast.

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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A brief review of phase contrast.

#1 Post by apochronaut » Sun Feb 23, 2020 8:16 pm

One of the easiest ways someone can add a new dimension to their basic BF microscope is to employ a different method of contrast in the substage, usually referred to as contrast enhancement. Dark field patches, rheinberg filters and various techniques to introduce oblique illumination have all been used with great effect over the years but no other technique has proven so precisely useful at such low cost as phase contrast.
Despite this, phase technology matured in an era of great development in the area of microscope contrast systems and quickly became a kind of poorer cousin to Polarizaton interference systems of substantialy higher cost and later , digitized and coherent illumination systems of even greater cost. For the amateur and non-professionals, many of whom are on a tight budget, phase contrast remains, the least expensive dramatic improvement one can generally make to a microscope system.


Phase contrast went through numerous developmental stages from the point of the postulation of it's theory to it's manifestation in a modern microscope. Part of that development , covering numerous ramifications and coating variations possible has all but been lost. The envisioned potential and experimental designs of the early proponents of phase contrast are a far cry from the 3 objective prhase contrast and condenser set available for an Amscope at 381.99. Modern basic phase contrast add ons make use of just a slice of what is possible . Fortunately, phase is quite flexible and anyone who enjoys diy'ing can play with that flexibilty, just as the pioneers did.

The first thing to know is that with phase, just about everything happens in the objective, or rather in the diffraction plate in the objective. During the heyday of phase development, everyone had their own ideas of phase design, so every diffraction plate will have some characteristics that make it different from another. What this does, is it provides a great deal of precision optical engineering to play with at very low cost since there are a lot of old phase objectives available on the used market.

It is widely known that Fritz Zernicke invented phase contrast microscopy but Zernicke was a physicist, not a microscope designer. His work was based on theories evolved by the British microscopist and physicist Julius Rheinberg and independently A.E. Conrady. Zernicke used their discoveries to further his research into perfecting the analysis of defects in complex optical systems.C.R. Burch used the Zernicke system in 1934 to analyze the defects in concave optical mirrors. However, Zernicke had secured patents and licensed those patents to Zeiss. It was Kohler and Loos of Zeiss ,that took the theory to the next stage. In 1941 and 42, photographs taken through a Zeiss microscope modified to utilize Zernike phase strips proved the value of the new system. It seems that the early Zernicke phase contrast system used linear phase modulation, more similar to Hoffman Modulation Contrast and it was through the combined efforts of a number of phase contrast researchers from 1941 or so on , that the modern concept of phase contrast as a condenser diaphragm coordinated to a matching annular diffraction plate took place. During the 1940's, a great deal of energy was put forth refining the concept and simplifying the hardware at about 7 companies worldwide, resulting in numerous finely tuned systems of phase contrast existing by 1950. It is interesting to note that by far the most expansive and user friendly system at that time was made by Officine Galileo, an Italian optical firm.
Phase Contrast is sometimes referred to as Zernicke phase contrast. Zernicke realized and patented the principal but in fact did very little to design any of the systems. By the late 40's there was a great deal of interest in the potential of phase microscopy and alternative designs emerged. One wss devised by a colleague of Georges Nomarski, Maurice Francon. In fact , Nomarski authored some papers on phase contrast. Francon's phase contrast microscope could utilize standard BF microscope optics but required a relfecting glass semisphere to be positioned between the slide and the objective. It is probable that tradition of referring to phase contrast as Zernicke phase contrast, results from the existence of alternative systems that did not rely on Zernicke's patent.

In the ensueing years since the great 1945 to say 1960, developmental era of phase contrast, numerous systems have evolved , come and gone. Some companies maintained a longer presence with some diversity of optical possibilities but by and large, phase systems are ubiquitously medium contrast and of the dark type, nowadays.
Numerous research papers and chapters of books describe the ability of specific phase systems to reveal detail in specific samples when another can not. Phase is not one method. It is a continuum and still waiting to be explored.

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Re: A brief review of phase contrast.

#2 Post by MicroBob » Sun Feb 23, 2020 8:36 pm

Hi Phil,
very interesting post, thank you! It is interesting to read that Officine Galileo offered such a good phase contrast system as they don't seem to have made many microscopes. Officine Galileo was founded by the well known Giovanni Battista Amici, one of the people who worked successfully in microcope development.

At least PZO and Zeiss Jena offered variable phace contrast systems.

Bob

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daruosha
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Re: A brief review of phase contrast.

#3 Post by daruosha » Sun Feb 23, 2020 9:17 pm

Fascinating. Thank you.
Would you share some of your knowledge on relief phase contrast illumination as well? Does it work with all phase contrast objective or it requires special type of phase plate/optical design?
Daruosh.

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Re: A brief review of phase contrast.

#4 Post by apochronaut » Sun Feb 23, 2020 9:36 pm

By 1949, Officine Galileo produced a Universal phase contrast binocular microscope with a pancratic achromatic aplanatic 1.40 condenser, carrying the moveable phase diaphram, with a built in illuminator below the condenser. The condenser was variable from .16 to 1.4. and only one annular diphragm was necessary because it changed it's dimensions when the condenser was varied. The objective complement was: dark contrast, 10.5X .28, 25X .65 and 52X .88 dry acromats ; 100X 1.30 oil achromat ; bright contrast, 100X 1.30 achromat oil . The clincher in the system, is that they appear to have invented the integrated bertrand lens.

F. Koristka had a version of that but I think you had to put in a different eyepiece to use it and also, they had a 100X 1.20 water immersion dark phase objective and a 100X 1.32 oil fluorite dark phase objective. Pretty nice stand too.

Zeiss-Winkel had 10X .25, 42X .85 achromat dark phase and a 90X 1.30 oil achromat all dark only.

Zeiss Jena had 10X .25, 20X .45, 42X .65 and a 90X 1.25 oil dark phase. Both companies used a separate phase telescope. Jena also supplied a pancratic condenser on the Lumipan.

AO definitely held sway in terms of objective options, with 26 different objectives in 3 types and 3 contrast ranges: albeit in 4 achromat magnifications and rather pedestrian N.A.s; 10X .25, 20X .50, 43X .66 and 97X 1.25. The series 35 stand required a phase telescope.

Other companies, Wild, Bausch & Lomb, R & J Beck , C Reichert and Cooke Troughton and Simms used existing basic stands with 3 to 4 objective dark phase options .

C. Baker had a system with cruciform diaphragms and phase plates.

PZO hadn't shown up yet.

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Re: A brief review of phase contrast.

#5 Post by apochronaut » Sun Feb 23, 2020 9:49 pm

daruosha wrote:
Sun Feb 23, 2020 9:17 pm
Fascinating. Thank you.
Would you share some of your knowledge on relief phase contrast illumination as well? Does it work with all phase contrast objective or it requires special type of phase plate/optical design?
That's the thing about phase contrast. There are so many options, yet it has become pretty dumbed down in most modern offerings.
The way I have been anle to get relief phase contrast is by using a combination of oblique and phase. I play around a lot with some hits and misses.
Using an illumination beam offset as well as oblique at the condenser has some value. I always use precisely aligned condenser diaphragm and diffraction plates if they are from the same system but when an objective and annular diaphragm are from separate mfgs., strange things can happen.

I also don't always look for a typical background colour. Some of my better resolved imaging is with slightly mis-matched systems which had a brown background.

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Re: A brief review of phase contrast.

#6 Post by Rossf » Sun Feb 23, 2020 9:52 pm

Very interesting Apochronaut for a phase enthusiast like me-and all the other input from everyone-even though modern phase is very precise and engineered for ease of use, I can see a lot of modern systems don’t seem to have a particular “sweet spot”-definitely improving contrast for most things but not like some of the older variants that say did a particularly good job at cillia or flagellates or mobile rod bacteria lined up like train carriages (Choo!Choo!) Nikon’s Bright Mediums where good for that-I’ve seen a few photos of some of the weird variants A.O used-some looked a bit like oblique-all very different-I still think a great development along the way was when Zeiss for example could use the one annulus for 3 magnifications (16,25,40x) making thing way simpler-It’s probably a good thing that true variable phase contrast isn’t on modern scopes-I’d never eat or got to bed!
Regards ross

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Re: A brief review of phase contrast.

#7 Post by apochronaut » Sun Feb 23, 2020 10:46 pm

Yes, simplifying phase has been of value and there is no doubt that utilizing better colour corrected objectives has been a benefit. Yet, I lament the fact that the possible diversity and specificity of phase is pretty much gone . AO, already by 1942 was experimenting with designs for a more variable phase system, where the phase and amplitude could be continuously modulated along a broad continuum. They eventually marketed it as the Polanret( polarization and retardation) system. As such it was an early competitor to D.I.C.
I have come across two such systems and they are rare and expensive.

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Re: A brief review of phase contrast.

#8 Post by microb » Mon Feb 24, 2020 3:55 am

https://patents.google.com/patent/US3658405

Citation given in patent:

Journal of the Optical Society of America, vol. 37, Sept. 1947, pages 726-730, H. Osterberg article “The Polanret Microscope"

Quote from patent:

"These prior proposals have found little practical application, as their manufacture is difficult or expensive. From among these devices the "polan ret" system developed by H. Osterberg is the most versatile. However, this system has, in the back focal plane of the microscope objective, a phase plate consisting of zonal polarizing filters which are very difficult to manufacture commercially. In this system the phase difference between the direct and diffracted beams is varied by rotation of a polarizer placed before the polarizing zonal plate and the intensity ratio of these beams is changed by rotation of an analyzer located be hind this zonal plate."
Last edited by microb on Mon Feb 24, 2020 5:16 am, edited 1 time in total.


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Re: A brief review of phase contrast.

#10 Post by MicroBob » Mon Feb 24, 2020 8:10 am

Hi together,
in my view it would be interesting to compare the results of different phase contrast setups by means of a more or less standardized object. To be able to stretch the testing out a bit it would be nice to be able to find a permanent mounted standard. What could this be? A piece of PE film mounted in canadabalsam?
I could contribute PZO variable phase (all objectives), Zeiss Jena (All objectives, have to repair and adjust the pancratic condenser :? ) and Zeiss West 160mm (few objectives), perhaps Zeiss Winkel (if compatible with later condernser annulli).

Bob

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Re: A brief review of phase contrast.

#11 Post by apochronaut » Mon Feb 24, 2020 12:31 pm

The variables would be hard to control.
For any given sample, there is an ideal phase set up. That is what makes systems that are capable of altering the functional variables of wave retardation and absorption incrementally or continuously, so valuable for a diversity of microscope subjects.
Research seemed to stall once D.I.C. became the force of nature, although obviously up to a certain point in time there were some good systems out and about.
Maybe some young enthusiast engineer in China will set to work on it and extract the base microscope industry out of the doldrums of " dark medium phase". It's not even called dark phase anymore; it's just phase....... it's like a toolbox with one tool in it : a medium sized slot head screwdriver.

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Re: A brief review of phase contrast.

#12 Post by microb » Mon Feb 24, 2020 4:16 pm

What about all the techniques with stacking different LCD displays together:

https://phioptics.com/technology/

http://light.ece.illinois.edu/wp-conten ... E_SLIM.pdf

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Re: A brief review of phase contrast.

#13 Post by apochronaut » Mon Feb 24, 2020 6:03 pm

apochronaut wrote:
Sun Feb 23, 2020 8:16 pm
For the amateur and non-professionals, many of whom are on a tight budget, phase contrast remains, the least expensive dramatic improvement one can generally make to a microscope system.
Hmmm. guess I would have to sell a portion of my 150 year old hard wood lot for one of those.
They say, phase microscope, not Bright Medium phase, or B-Minus phase, so presumably the programming is adjustable to whatever phase system is at hand, not just the kleenex box dark phase? The systems are a quantitative use of an image produced by the use of phase but the nature of the phase stays the same. Can they add or subtract , as in deduce information that isn't there; the kind of information a different phase diffraction plate would provide? If you have only one type of diffraction plate , for instance dark phase, the nature of the phase type stays the same with those systems, it just gets iinterpreted with some assistance from electrons, I would guess..
The systems I doubt could be used to predict an image that would be generated by a phase system of other conjugate and complementary area variables than those moderating the current set of photons. Maybe a Polanret would be a better buy= more original information.

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Re: A brief review of phase contrast.

#14 Post by KD Arvidsson » Mon Feb 24, 2020 9:18 pm

Thanks for interesting reading😊//KD
Microscope Nikon Labophot 2
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Westcoast of Sweden.
https://www.youtube.com/channel/UCjsgbq ... dyl2x0Atpw

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