Phase Contrast Question

Here you can discuss different microscopic techniques and illumination methods, such as Brightfield, Darkfield, Phase Contrast, DIC, Oblique illumination, etc.
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Red_Green
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Joined: Fri Jun 05, 2020 9:38 pm

Phase Contrast Question

#1 Post by Red_Green » Sun Feb 21, 2021 3:45 pm

G'day. I have a 30% off coupon for Amscope and was thinking of either upgrading to plan objectives (4x, 10x, 20x, and 40x) or getting the phase contrast kit.

I was wondering with phase contrast. I really hate the look of the halos I see in pictures taken with phase. Is there anyway to dial down the amount of halo you get with phase or at least anyway to dial them down in post processing of images taken?

Or should I just stick to upgrading to plan objectives?

Every time I buy something from Amscope I get a 15% off coupon. Which means if I buy 4 objectives I will end up with more coupons which will be 60% off which will then just make me want to buy more things.

Greg Howald
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Joined: Tue Oct 20, 2020 6:44 am

Re: Phase Contrast Question

#2 Post by Greg Howald » Sun Feb 21, 2021 4:08 pm

At lower magnification levels I have read on the forum that plan objectives don't seem to make a great difference in what is seen in your scope. I have them in 4, 10, and 40x. I don't see much difference and seldom use them. There are differences of course and that can be measured. Whether you can detect much difference with the limitations of human vision is another matter and must be based upon what is personally observed.
I also don't think you can avoid halos in phase contrast.
If it were my money I would go with phase contrast mounted on a turret, but that's just my opinion based solely upon preference.
Greg

PeteM
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Location: N. California

Re: Phase Contrast Question

#3 Post by PeteM » Sun Feb 21, 2021 4:44 pm

Only the central vision of our eyes is sharp. Our brains make up a sharp image through a variety of techniques (scanning the image with eye movements, continuously focusing, etc.). So a regular achromat objective can look pretty good at low magnification.

Typical cameras aren't so forgiving. If the edges are out of focus, they'll record it. While there's a bit more depth of field at low magnifications - to my mind plan objectives make sense for serious photographers at every magnification. Someone else with a better knowledge of focus stacking might know if plan objectives also make for better stacks?

With something like pond critters both eyes and cameras have trouble keeping them in focus as they move up and down in the thin bit of space under a cover slip. A regular achromat or a phase acromat at, say, 20x magnification can make sense, even taking movies. One just has to track and focus them as best as possible - and prepare a just-right sample with enough water for them to live and move and a thin enough layer of that water to keep them somewhat constrained.

The halos pretty much come with phase contrast, though I understand there are some new research techniques that minimize this. You also lose a bit of resolution in brightfield. Some don't like darkfield or can't be used with DIC or polarization. But since so many live cells are essentially bags of water - phase contrast really is useful. Traditional work-arounds to gaining contrast include staining, phase contrast, polarization, darkfield, oblique, Hoffman modulation contrast, and of course DIC at a higher price.

I'd be surprised if AmScope lets you double, triple, and quadruple up on 15% coupons - eventually getting 60% off an item. Are you sure this is possible??

apochronaut
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Re: Phase Contrast Question

#4 Post by apochronaut » Mon Feb 22, 2021 3:51 pm

There are 5 fairly controllable phenomena with phase contrast that can affect the image quality greatly.
1) the evenness, focus and colour temp. of the background illumination $
2) the type of phase contrast system. $$
3) the thickness of and type of sample. no $
4) the colour correction level of the objectives being used. $$$
5) the critical alignment of the system prior to use. no $

Two of those parameters are cost free and involve only experience skill and precision to execute. Careful attention to sample preparation can reduce the halo some. Alignment improves resolution and detail. This includes the aignment of the stand as well as phase alignment. Having the phase condenser diaphragm and phase plate aligned are one thing but if the filament/emitter, collimator lens, field diaphragm, condenser lens center, objective and head are like a spirochaete: image quality will suffer.

The quality of the illumination /condensing system is usually fixed in the stand, although some have the capacity for upgrading. If the microscope is an off the shelf inexpensive Chinese or Indian model, the likliehood of the phase condenser being the same condenser as is used for BF is high. This is not normal for high quality phase systems, where a specific condenser is designed for phase. Phase does not require a high N.A. condenser. An N.A. below 1 is ideal but also an aspheric or achromatic version is called for, due to the demands of the illumination field. A 1.25 abbe oil condenser that does double duty as a dry phase condenser is not ideal and will more than likely contribute to a reduction in image quality.

Most phase systems offered for stencil microscopes are positive or dark phase and most likely of medium contrast. Over time, this has proven to be the most popular type and where only one type of phase system is offered, that is it. It is not always the best for every microscopic subject. There are a few of the stencil systems out there that offer a negative or bright phase contrast system as well. This involves just additional objectives, not an additional condenser. More established companies offer multiple systems. With many subjects, the halo effect is reduced dramatically with a change in the phase type and less obvious with objectives of a higher colour correction.

All phase systems offered for stencil microscopes that I have encountered are achromat or plan achromat. With systems that I have used, the image quality of planfluorite or planapo phase is dramatically superior. Since most of the stencil scopes being made use clones of either the Olympus, Nikon 45mm or Nikon 60mm optical systems, it may be possible to in future improve a basic phase system by acquiring more elite phase objectives from a compatible Japanese system or even possibly a better phase condenser in the achromat category.
I did this with a Microstar IV, which was originally configured for a 4 objective dark medium planachro system only. It does use a .90 achromat/aplanat however. There were other phase objective options in the planfluor and planapo categories for Austrian microscopes such as the Diavar II and possibly the Polylite, which also sold in N.America. Several of these showed up at decent enough prices off and on , so that I was able to improve the phase performance of the Microstar quite a bit and for not a huge amount (500.00 for 3 objectives : 2 planfluor apo and 1 planapo).The halo with those better corrected objectives is quite low or at least not so bothersome. The resolution very high.
Last edited by apochronaut on Tue Feb 23, 2021 2:40 pm, edited 1 time in total.

BramHuntingNematodes
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Location: Georgia, USA

Re: Phase Contrast Question

#5 Post by BramHuntingNematodes » Tue Feb 23, 2021 5:02 am

Not sure what your use case is but I would go with the phase system. Rather than an incremental correction to the bright field image it transforms phase differences into amplitude differences it's like a kind of magic. Also, the condenser can be used for COL and sometimes darkfield.

Halos are useful sometimes in further enhancing contrast at boundaries and edges. This is very apparent when looking through the eyepieces at a live specimen but not easily captured with a casual photo. It has a captivating appearance and the contrast is sometimes electric!

You can adjust some things to control them a bit. You like making tons of tiny adjustments to get the perfect image don't you? Sure you do. That's half the fun.
1942 Bausch and Lomb Series T Dynoptic, Custom Illumination

Red_Green
Posts: 20
Joined: Fri Jun 05, 2020 9:38 pm

Re: Phase Contrast Question

#6 Post by Red_Green » Thu Feb 25, 2021 4:10 am

PeteM wrote:
Sun Feb 21, 2021 4:44 pm
I'd be surprised if AmScope lets you double, triple, and quadruple up on 15% coupons - eventually getting 60% off an item. Are you sure this is possible??
I tried to use the coupons today and neither of them worked and said they were invalid. They gave me two bogus coupons lol.

Assuming they did work, I think you would only be able to use one at a time.

I did message them about it and gave them screen shots and they apologized and said they would credit one of the 15% off coupons towards the order I left a review on though.

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