Relief Phase Contrast Microscopy - Article by Prof J. Piper in Microscopy and Analysis, vol 21, 2007.

Here you can discuss different microscopic techniques and illumination methods, such as Brightfield, Darkfield, Phase Contrast, DIC, Oblique illumination, etc.
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Hobbyst46
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Relief Phase Contrast Microscopy - Article by Prof J. Piper in Microscopy and Analysis, vol 21, 2007.

#1 Post by Hobbyst46 » Fri Oct 11, 2019 11:55 am

Wes wrote:
Thu Oct 10, 2019 10:42 pm
Speaking of illumination techniques I find relief phase contrast interesting
http://zeiss-campus.magnet.fsu.edu/refe ... 9-2007.pdf
Haven't yet had the patience to properly explore, maybe someone who has can chime in.
Motivated by this link, I found that article to be enjoyable reading. There are several points that, in my opinion, are worthy of consideration.
0. The method is essentially oblique illumination combined with PC.
1. The method can create different and richer, so to speak, images than conventional PC. The features of the results are summarized in Table 1 of the article. There are plus and minus points.
2. The method is seemingly very easy to implicate in ordinary PC microscopes, especially using a turret PC condenser (side note: Piper calls this condenser type a "Zernike phase contrast condenser"; perhaps, Prof Zernike influenced Zeiss to manufacture specifically turret PC condensers; Piper in his article used Leitz microscopes; whatever...). In the simplest way, rotate the turret to misallign the condenser annulus with the objective phase ring. Or combine an obliquely-located annular section with the condenser iris aperture to adjust the oblique PC illumination.

In my opinion, this method might be attractive to hobby microscopy, rather than to professionals. I suspect that relief PC images are not very appropriate for quantitative image analysis, at least because the background is not uniformly bright.

Two technical limitations that I see, for hobbyists who do not wish to fabricate specific opto-mechanical gadgets:

A - the relief PC needs much brighter light sources than ordinary PC (which, in turn, needs much brighter sources than brightfield).
B - some of the methods suggested by Piper are not easily feasible, because not all turret PC condensers have the iris aperture in series with a phase annulus. On my two different types of Zeiss PC condensers, for example, the iris aperture is installed in the BF, and the phase annuli see an empty opening (maximum aperture), which is fine for ordinary but not for relief PC.

So, Relief PC is added to my to do list.
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MicroBob
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Re: Relief Phase Contrast Microscopy - Article by Prof J. Piper in Microscopy and Analysis, vol 21, 2007.

#2 Post by MicroBob » Fri Oct 11, 2019 1:04 pm

Hi Doron,
Jörg Piper has done quite a bit in microscopy:
http://www.joerg-piper.de/

On relief-phase contrast:
http://www.joerg-piper.de/Relief-Phasen ... trast.html

His MIKROKOSMOS-Article on relief phase contrast:
https://www.zobodat.at/pdf/Mikrokosmos_96_4_0001.pdf

The images he shows with relief phase contrast are indeed quit contrasty, but also prone to artefacts. So like with oblique and DIC and with stacked images it is an advantage to know how to differentiate between object and artefact. I can imagine that is is easier to get a repeatable result than with oblique.
It might be interesting to experiment with phase stops that have polarizers on a part of their phase ring to make the effect adjustable.

Bob

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Re: Relief Phase Contrast Microscopy - Article by Prof J. Piper in Microscopy and Analysis, vol 21, 2007.

#3 Post by Hobbyst46 » Fri Oct 11, 2019 1:31 pm

Thanks Bob, a wealth of articles indeed! downloaded 6 articles and now, let's see what are the practical playthings for free or on the cheap!
The advantage of simple classical PC is that if you own the proper accessories, it works 100%. Without tweaking and black witchcraft 8-)
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Re: Relief Phase Contrast Microscopy - Article by Prof J. Piper in Microscopy and Analysis, vol 21, 2007.

#4 Post by MicroBob » Fri Oct 11, 2019 1:37 pm

Hobbyst46 wrote:
Fri Oct 11, 2019 1:31 pm
Without tweaking and black witchcraft 8-)
In my eyes this is the main disadvantage od oblique/COL. It reacts a lot on fine adjustments and is more an art or craft and not easily to reproduce.
On the other hand side: If one would use a high quality revolver condenser and equip it with 5 fixed oblique setting and demand from the user to only use it with fixed, specified objectives, it would be a lot more repeatable. And these are the accepted restrictions for the use on DIC!

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Re: Relief Phase Contrast Microscopy - Article by Prof J. Piper in Microscopy and Analysis, vol 21, 2007.

#5 Post by apochronaut » Fri Oct 11, 2019 4:52 pm

[/quote]
(side note: Piper calls this condenser type a "Zernike phase contrast condenser"; perhaps, Prof Zernike influenced Zeiss to manufacture specifically turret PC condensers; Piper in his article used Leitz to my to do list.
[/quote]

What is the date of the earliest phase contrast condenser that had a rotary carousel holding the annuli? For sure, they existed in 1948. Does anyone know of one earlier?

Regarding Zernike " influencing Zeiss" . Zeiss had forced labour camps. Zernicke was a Dutch Jew. duh!

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Re: Relief Phase Contrast Microscopy - Article by Prof J. Piper in Microscopy and Analysis, vol 21, 2007.

#6 Post by MicroBob » Fri Oct 11, 2019 6:04 pm

Hi Phil,
this is an interesting aspect! I looked in the internet and found:
-Frits Zernicke developed phase contrast in 1933 but Zeiss didn't make use of it
-He published the Van Critter-Zernike theorem in 1938
-The german Wehrmacht unearthed phase contrast in 1941 when looking for patents important for the war (Why phase contrast???)
-Zeiss put phase contrast into use in microscopy 1941 (really? how many sets?)
-His first wife died in February 1945
-He became member of the Royal Netherlands Academy of Arts and Sciences in 1946

I didn't find anything about what he did between 1938 and the end of war. Not much for somebody awarded a nobel prize! But maybe completely normal for an academic in the war time in the Netherlands.
It would be interesting to know what he did and how he survived as a jew or at least with jewish parents in the german occupied Netherlands.
It would also be interesting to know why Zeiss didn't make use of his patent (I think to remember that he actively offered it to them).
Pure ignorance? Too many war preparations to do? Unwillig to believe that somebody outside Zeiss company could develop something important? Unwilling to cooperate with an inventor of jewish background?

Bob

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Re: Relief Phase Contrast Microscopy - Article by Prof J. Piper in Microscopy and Analysis, vol 21, 2007.

#7 Post by 75RR » Fri Oct 11, 2019 6:36 pm

Horses mouth

"With the phase-contrast method still in the first somewhat primitive stage, I went in 1932 to the Zeiss Works in Jena to demonstrate. It was not received with such enthusiasm as I had expected. Worst of all was one of the oldest scientific associates, who said: « If this had any practical value, we would ourselves have invented it long ago »."


https://www.nobelprize.org/uploads/2018 ... ecture.pdf
Last edited by 75RR on Fri Oct 11, 2019 6:49 pm, edited 1 time in total.
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Re: Relief Phase Contrast Microscopy - Article by Prof J. Piper in Microscopy and Analysis, vol 21, 2007.

#8 Post by Hobbyst46 » Fri Oct 11, 2019 6:48 pm

75RR wrote:
Fri Oct 11, 2019 6:36 pm
Horses mouth

https://www.nobelprize.org/uploads/2018 ... ecture.pdf
Thanks 75RR. Glad to see that the side note was fairly correct. And the Lecture also states that phase contrast accessories were introduced by Zeiss in 1941. After several visits of Zernike in Jena. They were not enthusiastic about his invention initially...
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Re: Relief Phase Contrast Microscopy - Article by Prof J. Piper in Microscopy and Analysis, vol 21, 2007.

#9 Post by Hobbyst46 » Fri Oct 11, 2019 8:07 pm

MicroBob wrote:
Fri Oct 11, 2019 6:04 pm
...Frits Zernicke developed phase contrast in 1933 but Zeiss didn't make use of it
-He published the Van Critter-Zernike theorem in 1938
-The german Wehrmacht unearthed phase contrast in 1941 when looking for patents important for the war (Why phase contrast???)
-Zeiss put phase contrast into use in microscopy 1941 (really? how many sets?)
-His first wife died in February 1945
-He became member of the Royal Netherlands Academy of Arts and Sciences in 1946
I didn't find anything about what he did between 1938 and the end of war. Not much for somebody awarded a nobel prize! But maybe completely normal for an academic in the war time in the Netherlands.
Zernike was a leading scientist in several fields of mathematics (including statistics) and physics, phase contrast was just one (prominent) of his achievements. His close family were intelectuals, some of them mathematicians.
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Re: Relief Phase Contrast Microscopy - Article by Prof J. Piper in Microscopy and Analysis, vol 21, 2007.

#10 Post by 75RR » Fri Oct 11, 2019 9:11 pm

75RR wrote:
Fri Oct 11, 2019 6:36 pm
Horses mouth

"With the phase-contrast method still in the first somewhat primitive stage, I went in 1932 to the Zeiss Works in Jena to demonstrate. It was not received with such enthusiasm as I had expected. Worst of all was one of the oldest scientific associates, who said: « If this had any practical value, we would ourselves have invented it long ago »."


https://www.nobelprize.org/uploads/2018 ... ecture.pdf
MichaelG.'s second link (see below) would seem to show that Zernike wisely Patented his invention before visiting Zeiss in 1932

viewtopic.php?f=5&t=8166#p71615
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Re: Relief Phase Contrast Microscopy - Article by Prof J. Piper in Microscopy and Analysis, vol 21, 2007.

#11 Post by MicroBob » Sat Oct 12, 2019 2:36 am

MicroBob wrote:
Fri Oct 11, 2019 6:04 pm
as a jew or
Doron made me aware that Frits Zernike probably was not a jew. I tried to find this bit of information again in the internet and only found a google hit that mixed up two scientists in one search list entry.
Whatever religion he had: Nice he invented phase contrast for us!

Bob

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Re: Relief Phase Contrast Microscopy - Article by Prof J. Piper in Microscopy and Analysis, vol 21, 2007.

#12 Post by Wes » Sat Oct 12, 2019 8:30 am

In case anyone tries relief PC please share your results here. I tried something but so far it just looked rather bad.

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Re: Relief Phase Contrast Microscopy - Article by Prof J. Piper in Microscopy and Analysis, vol 21, 2007.

#13 Post by Hobbyst46 » Sat Oct 12, 2019 10:37 am

Hobbyst46 wrote:
Fri Oct 11, 2019 11:55 am
Two technical limitations that I see, for hobbyists who do not wish to fabricate specific opto-mechanical gadgets:
A - the relief PC needs much brighter light sources than ordinary PC (which, in turn, needs much brighter sources than brightfield).
B - some of the methods suggested by Piper are not easily feasible, because not all turret PC condensers have the iris aperture in series with a phase annulus. On my two different types of Zeiss PC condensers, for example, the iris aperture is installed in the BF, and the phase annuli see an empty opening (maximum aperture), which is fine for ordinary but not for relief PC.
To which, after having read a few more articles by J. Piper about the subject, I would add:
C the advantage of the so-called relief phase contrast depends on specific properties of the optics in use (for example: on the amount of corrections of the objective {achromat/plan etc} and its magnification). By contrast, phase contrast (pun) has a definite advantage over brightfield at all mags.
D - Piper presented images taken with interference filters of a narrow bandwidth (8-9nm). Although monochromatic light can improve resolution, like in limitation A above, it needs very bright light sources. My own 10W white LED + narrow bandwidth green filter failed to show a clearly visible image through the binocular eyepieces.
Still, might give a chance to partial rotation of phase condenser annuli to create oblique PC.
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Re: Relief Phase Contrast Microscopy - Article by Prof J. Piper in Microscopy and Analysis, vol 21, 2007.

#14 Post by MicroBob » Sat Oct 12, 2019 12:55 pm

The combination phase annuli and aperture is really not very common. It might be part of a variable phase contrast set where an outer phase annulus can be covered by the aperture to use an inner annulus instead. The Zeiss Jena pancrativ condenser with it's single phase annulus could be used this way as it is for variable phase contrast. I have a PZO variable phase kit, but have to look whether there is an aperture available.

A beginning relief effect will be obtainable by blocking out just a bit of the phase annulus, e.g. 25%. This wouldn't reduce the brightness so much. In your Zeiss West phase condenser the gap between cover disc and rotating disc might be wide enough to get to the active condenser port with a strip of aluminium foil or paper. The phase annule themselves are quite fragile (paint on glass) so I wouldn't come close to them if not necessary.

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Re: Relief Phase Contrast Microscopy - Article by Prof J. Piper in Microscopy and Analysis, vol 21, 2007.

#15 Post by 75RR » Sat Oct 12, 2019 2:19 pm

I really don't think that Relief Phase is a viable illumination technique.

I don't think this is Phase without a Halo, which I agree sounds attractive. The reason there is no Halo is because there is no Phase.

This is Oblique using the Phase ring in Phase objectives - I think calling it Relief Phase is a misnomer.

Phase Ring Oblique would be more accurate but even then I am not convinced about the appropriation of the word Phase in this context.
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Re: Relief Phase Contrast Microscopy - Article by Prof J. Piper in Microscopy and Analysis, vol 21, 2007.

#16 Post by mnmyco » Fri Oct 18, 2019 5:37 am

I have ended up with two turret condenser bodies for my AO series 10 and 20 microscope. Once I get them both in working order (need another trinocular and series 20 lamp cord and socket) I will try to create a much more controlled test of this “relief phase contrast”.

MNMyco

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