can Phase contrast kit work WITHOUT Köhler ???

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chrisimbee
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can Phase contrast kit work WITHOUT Köhler ???

#1 Post by chrisimbee » Sat Apr 10, 2021 10:11 am

Hello ,

I have a big question for the one who own a phase contrast kit :

Could try to see if it still work when your microscope isnt set in Köhler illumination setting ?

I cant understand why I could get my x40 Ph LOMO works perfetly with no problm and not do the same with the LOMO Ph x10 ??? (of course I made a different annulus phase matching the ring on the phase plate).

I know about the theory and optics of phase contrast and interferences production but still it puzzles me a lot.

I even bought another LOMO x10 but this time with the same conception as the x40 LOMO that works well. I wondered if my x10 wasnt designed for a specific setup a suggested in another post.

And I it works could post some photos showing the difference in quality of image if there are any ?

Thanks a lot
Chris
microscope Olympus BH2-BHTU+epifluo RFC @ 470 nm
Zeiss neofluar x16Ph, x40Ph, x100 oilPh
LOMO Ph x10 X20 X40 X90oil
Olympus SPFl x2
Olympus SPx20, SPx40 SPx100
camera astro ZWO ASI 120MM (n&b) et ZWO ASI 120 MC (colour)
Nikon D3100

chrisimbee
Posts: 105
Joined: Fri Mar 19, 2021 5:34 pm

Re: can Phase contrast kit work WITHOUT Köhler ???

#2 Post by chrisimbee » Sat Apr 10, 2021 1:54 pm

a piece of information :

At my lunch break I tried a few things : I tried to focus the annulus with a standard x20 and its the same problm : i cant get an image ;
With a x60 i can get an image but only partially..

So it could mean its probably related to the focal lenth ; i'll try the calculations.
microscope Olympus BH2-BHTU+epifluo RFC @ 470 nm
Zeiss neofluar x16Ph, x40Ph, x100 oilPh
LOMO Ph x10 X20 X40 X90oil
Olympus SPFl x2
Olympus SPx20, SPx40 SPx100
camera astro ZWO ASI 120MM (n&b) et ZWO ASI 120 MC (colour)
Nikon D3100

Red_Green
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Joined: Fri Jun 05, 2020 9:38 pm

Re: can Phase contrast kit work WITHOUT Köhler ???

#3 Post by Red_Green » Sat Apr 10, 2021 4:17 pm

I own the Amscope phase contrast turret kit and it works perfectly without Köhler. So I would imagine that any kit could, although keeping in mind that vintage kits and such were invented back when microscopes still used tungsten lighting which is why Köhler was needed in the first place and now in modern times tungsten lighting is not used so therefore Köhler is not really needed but rather more of an Aesthetics and nostalgia thing according to my research.

With that said. I kind of wish my microscope had Köhler or some form of lighting control with the turret as there is no way to really control light with the amscope phase kit. Which is fine because the view and results is spectacular anyway. Just kind of a nit picky thing for me.

PeteM
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Re: can Phase contrast kit work WITHOUT Köhler ???

#4 Post by PeteM » Sat Apr 10, 2021 6:39 pm

If you don't already have one, you might want to get a centering or phase telescope to adjust things so that the phase annulus and the objective phase ring are coincident - with no light sneaking around your DIY phase rings.

dtsh
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Location: Wisconsin

Re: can Phase contrast kit work WITHOUT Köhler ???

#5 Post by dtsh » Sat Apr 10, 2021 7:29 pm

chrisimbee wrote:
Sat Apr 10, 2021 10:11 am
Hello ,

I have a big question for the one who own a phase contrast kit :

Could try to see if it still work when your microscope isnt set in Köhler illumination setting ?

I cant understand why I could get my x40 Ph LOMO works perfetly with no problm and not do the same with the LOMO Ph x10 ??? (of course I made a different annulus phase matching the ring on the phase plate).

I know about the theory and optics of phase contrast and interferences production but still it puzzles me a lot.

I even bought another LOMO x10 but this time with the same conception as the x40 LOMO that works well. I wondered if my x10 wasnt designed for a specific setup a suggested in another post.

And I it works could post some photos showing the difference in quality of image if there are any ?

Thanks a lot
Chris
While Köhler helps it isn't an absolute necessity. What matters most is having the condenser centered, centering the annulus in the condenser, and having the condenser at the right height. Obviously one needs to be using a annulus that is compatible with the objective.

Do you have a phase telescope or bertand lens? Do the two rings line up?


Edit: I took a couple of images to show the effect of ring misalignment. Note that I'm not suggesting mine is an example of a well aligned PC microscope, it isn't, but the phase rings still show the result.

Here's an image with the bertrand lens in place.
PC1.jpg
PC1.jpg (96.09 KiB) Viewed 6133 times
And the subsequent image is produces.
PC1_s.jpg
PC1_s.jpg (136.7 KiB) Viewed 6133 times

Now a shot of the bertrandl lens with the rings out of alignmnet.
PC2.jpg
PC2.jpg (55.06 KiB) Viewed 6133 times
And the resulting image
PC2_s.jpg
PC2_s.jpg (108.09 KiB) Viewed 6133 times

deBult
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Re: can Phase contrast kit work WITHOUT Köhler ???

#6 Post by deBult » Sat Apr 10, 2021 8:26 pm

No Kohler required: e.g. the Olympus CH2 has perfect phase with 10 and 40* using the CH2 phase kit.
The CH2 has critical illumination only (well I stand corrected: not critical but a strong diffuser after the lamp, see Apochromat’s comment below)

Question: did you open up the condenser diaphragm 100% (some phase condenser do this automatically) when testing the 10* ?
Last edited by deBult on Sun Apr 11, 2021 4:55 pm, edited 2 times in total.

chrisimbee
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Re: can Phase contrast kit work WITHOUT Köhler ???

#7 Post by chrisimbee » Sun Apr 11, 2021 9:38 am

Thank you so much for your answers.

question : are the photos taken with the X10 objective ?

Its great that citical illumination can give phase contrast as its never mentioned anywhere.

ive been successfull so far with my DIY phase contrast with a LOMO x40. My homemade phase annulus works fine but it can get better when i will print it out instead of drawing it. The reasonnable size of it (compared to a x10 annulus) is rather easy to make and center by hand.

Working with the x10 is another matter but I think I understood why : the spherical abberation of my Abbe condenser makes things difficult for a x10 and x20 objectives : only the central region of the field at the focal plane est flat : that means that when I insert my annulus into the condenser only the central stop blocks the ligh. The outer part of the annulus isnt sharp and looks gray (the central zone looks deep black). And because of some geometrical optics reasons I havent figured out yet (i think its linked to NA of the objectives) its easier with the x10 to get a larger flat zone at the focal plane of the condenser.

I expect that a proper phase condenser must have an aplanatic condenser to allow a proper position of the annulus and enough precision to center it... but still i'll carry on my DIY unitl i can get my hands on a not too expensive phase contrast kit

Thnks a lot for your help
microscope Olympus BH2-BHTU+epifluo RFC @ 470 nm
Zeiss neofluar x16Ph, x40Ph, x100 oilPh
LOMO Ph x10 X20 X40 X90oil
Olympus SPFl x2
Olympus SPx20, SPx40 SPx100
camera astro ZWO ASI 120MM (n&b) et ZWO ASI 120 MC (colour)
Nikon D3100

dtsh
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Location: Wisconsin

Re: can Phase contrast kit work WITHOUT Köhler ???

#8 Post by dtsh » Sun Apr 11, 2021 3:29 pm

Photos taken with 10x eyepieces (afocal with cellphone)

apochronaut
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Re: can Phase contrast kit work WITHOUT Köhler ???

#9 Post by apochronaut » Sun Apr 11, 2021 4:09 pm

There are more types of illumination than just critical or Köhler. Those are just two extremes related to how focused or defocused the illumination source is.
Critical illumination requires that the illumination source be very broad and quite uniform in illumination intensity. It was the illumination source of choice when the sky, gas flame or ribbon filament were used because the illuminator could be focused at the object plane and provide a decently uniform illumination field over the less than 15mm f.o.v. used in those days. It would be odd for any microscope with a coil or wire filament illumination source to use critical illumination because it would be impossible to magnify such a filament sufficiently in order to cover the field, especially a modern wide field.
Köhler is the opposite of critical. The filament is completely defocused at the object plane, providing a broad illumination field .

In practice, many microscopes use modified versions if those two extremes: intermediary between the two, since there are no laws that a microscope need be one or the other. Köhler is a much overused terminology currently, largely due to too many inexperienced microscopists assuming that Köhler is a necessity in order for a microscope to have a good illumination system. All a Köhler design does is provide assurance that the illumination system performs in a certain way, so it makes the operation of the system easier. Many alternative designs use a ground glass just forward of or integrated with a collimating lens, which helps to diffuse the filament image. Some use a complicated group of lenses, which does a similar function, so there are a number of proprietary "köhler type" designs that work similarly. Even a full on critical illumination design with diffusers will work as well. Led illumination has further complicated the situation by in some cases providing a wide flat illumination field with minimum required collimation, so a critical illumination system in effect.

Köhler makes setting up for phase easy, if it is integrated into the scope but any system that provides an even flat illumination field conjugate with the subject plane will do. The focal point, correction level and colour correction level of the condenser on top of that is a highly critical factor in phase.
Last edited by apochronaut on Sun Apr 11, 2021 8:20 pm, edited 1 time in total.

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patta
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Re: can Phase contrast kit work WITHOUT Köhler ???

#10 Post by patta » Sun Apr 11, 2021 6:16 pm

The Køhler issue seems to have been well answered above.

So, my guess is that the annulus of the 10x is so tiny and need to be centered so very precisely, that it is pretty difficult to get it in the right place manually ; as dtsh images. For the 10x, guess you will need like 0.2mm centering precision. Doable, but not easy! Also a tiny miscollimation between objective and condenser may make things harder I imagine.

If phase is working for the 40x, the condenser aberrations should give no problems for the 10x, since has smaller aperture.
The "foggy" impression when looking with Bertrand lens/centering eyp/no eyepiece, in my experience, come usually from the objective, not from the condenser.

I have done several homemade filter-tray-annulus, and found that best is to make the annulus slightly off-center, so the filter can be rotated to find the optimal centered position, coupled with some delicate swinging of the filter tray (which ain't perfectly centered). 0.5 mm centering comes easy; 0.2 mm, harder. Also, I make the annulus much thinner than the objective ring. For your 10x, it will be hair-thin!

Edit
I see from your previous post, you don't have the filter tray on the condenser.. that will make things harder to center. But no despair, Japanese masters are perfectly capable of moving things by half micron steps, bare hands. Also, the condenser should be "in focus", when you image and also same place when you measure what diameter draw the annulus. You may use the 40x to position the condenser.
To see the annulus in focus, the 10x objective will need a different focusing from your centering eyepiece, because its longer focal length, its aperture plane is in different place than the 40x. - don't move the condenser, nor the microscope focus; move the centering eyepiece!

apochronaut
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Re: can Phase contrast kit work WITHOUT Köhler ???

#11 Post by apochronaut » Sun Apr 11, 2021 8:15 pm

I should have added that the condenser is never oiled for phase. It works at sub 1.0 N.A. but arrives at that differently than DF.

chrisimbee
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Re: can Phase contrast kit work WITHOUT Köhler ???

#12 Post by chrisimbee » Mon Apr 12, 2021 8:01 am

Hello guys,

thanks again for your answers but I think I've found the reason WHY i cant make my x10 work like my x40..
obj LOMO ph x40.jpg
obj LOMO ph x40.jpg (113.36 KiB) Viewed 5961 times
obj LOMO ph x10  b.jpg
obj LOMO ph x10 b.jpg (133.36 KiB) Viewed 5961 times
As you can see they indeed are the same BRAND but there arent the same SERIES!!! and it makes the whole difference..

Why ?

because when I check the position of the phase plate in each of these objective I could see they were not at the same place at all!!!!

the x40 has its phase plate a bit after the back focal plane and thats why I can make it work fine ; I even made several simulation with an optic soft on my phone and it show how easy it is to set the phase annulus .

The x10 has its phase plate BEFORE the back focal plane and in that case the only place that works is to put the phase annulus in the condenser a bit BEFORE its focal plane. And unless i have a proper phase condenser its almost impossible by hand.

So it seems that theese 2 objectives cant work on the same condenser OR the x10 has a internel defect when it was mounted. It works fine in BF but not in PC unlike the x40 that works fine in both case.


If you wonna chek your phase plate its easy :

I use a jewelers magnifying glass (i dont know the english word) x4 for it.

1- hold your phase objective in one hand , the jewelers thingy at the eye and aim for an object located at an infinite distance ( landscape etc)

2- then you can see sharply in order : the frontal lens rim (pupille) ; then the reversed image of the landscape ; and then at last the phase plate (a gray ring).


In all books and websites they say that it's supposed to be located at the back focal plane (or slightly after)...

Could some of you chek if they see a similar difference between their x40 and x10 and others ?

thanks

Chris
microscope Olympus BH2-BHTU+epifluo RFC @ 470 nm
Zeiss neofluar x16Ph, x40Ph, x100 oilPh
LOMO Ph x10 X20 X40 X90oil
Olympus SPFl x2
Olympus SPx20, SPx40 SPx100
camera astro ZWO ASI 120MM (n&b) et ZWO ASI 120 MC (colour)
Nikon D3100

chrisimbee
Posts: 105
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Re: can Phase contrast kit work WITHOUT Köhler ???

#13 Post by chrisimbee » Mon Apr 12, 2021 8:03 am

sorry ; the previous photos didnt show what i meant
obj LOMO ph x40 b .jpg
obj LOMO ph x40 b .jpg (121.63 KiB) Viewed 5961 times
obj LOMO ph x10.jpg
obj LOMO ph x10.jpg (130.62 KiB) Viewed 5961 times
microscope Olympus BH2-BHTU+epifluo RFC @ 470 nm
Zeiss neofluar x16Ph, x40Ph, x100 oilPh
LOMO Ph x10 X20 X40 X90oil
Olympus SPFl x2
Olympus SPx20, SPx40 SPx100
camera astro ZWO ASI 120MM (n&b) et ZWO ASI 120 MC (colour)
Nikon D3100

jmp
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Location: Texas

Re: can Phase contrast kit work WITHOUT Köhler ???

#14 Post by jmp » Mon Apr 12, 2021 9:11 pm

LOMO objectives with a B.И. label (and white ring) are Water Immersion objectives. The markings of that 10x/0.25 objective that you show in that last image say B.M. (not B.И.) and have the white ring, so I wonder if that's a water immersion objective and not a dry objective like the 40x that you show above it. The 10x says SWIFT, not LOMO though I recall seeing somewhere that some LOMOs were rebranded as SWIFT at some point.

Come to think about it, I don't recall ever seeing a 10x/0.25 water immersion objective (LOMO or otherwise), so I'm probably way off.

viktor j nilsson
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Re: can Phase contrast kit work WITHOUT Köhler ???

#15 Post by viktor j nilsson » Mon Apr 12, 2021 10:27 pm

BM = bright medium, aka negative phase contrast.

jmp
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Location: Texas

Re: can Phase contrast kit work WITHOUT Köhler ???

#16 Post by jmp » Mon Apr 12, 2021 11:09 pm

BM = bright medium, aka negative phase contrast.
Thanks, that makes sense.

chrisimbee
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Re: can Phase contrast kit work WITHOUT Köhler ???

#17 Post by chrisimbee » Tue Apr 13, 2021 5:02 pm

hello :

sorry but i'm a bit dumb in CP : is bright médium = negative contrast OMG!! that explains why everything seemed so weird : the cheek ceels with my x40 are dark blue with their nucleus dark and with the x10 it looks the opposite in a golden light!! I couldnt understand why the vision with my x10 was so different!!

In fact it works pretty well but its a Ph- !!

thank you so much!!
microscope Olympus BH2-BHTU+epifluo RFC @ 470 nm
Zeiss neofluar x16Ph, x40Ph, x100 oilPh
LOMO Ph x10 X20 X40 X90oil
Olympus SPFl x2
Olympus SPx20, SPx40 SPx100
camera astro ZWO ASI 120MM (n&b) et ZWO ASI 120 MC (colour)
Nikon D3100

chrisimbee
Posts: 105
Joined: Fri Mar 19, 2021 5:34 pm

Re: can Phase contrast kit work WITHOUT Köhler ???

#18 Post by chrisimbee » Tue Apr 13, 2021 5:12 pm

and i forgot to say :

not only my x10 Ph- works well but even in the same way I use the x40 : without phase condenser and i put the phase annulus the same way I do with the x40 and Not in Köhler illumination. Of course in hte end I bought the Lomo phase condenser en ebay and i'm waiting for it to see how it"ll work and the difference i'll get with both way of proceeding.

Chris
microscope Olympus BH2-BHTU+epifluo RFC @ 470 nm
Zeiss neofluar x16Ph, x40Ph, x100 oilPh
LOMO Ph x10 X20 X40 X90oil
Olympus SPFl x2
Olympus SPx20, SPx40 SPx100
camera astro ZWO ASI 120MM (n&b) et ZWO ASI 120 MC (colour)
Nikon D3100

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