Disadvantages to Phase Contrast objectives?

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Goomba
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Disadvantages to Phase Contrast objectives?

#1 Post by Goomba » Wed Feb 26, 2020 3:04 pm

Hi all!
Silly question.
I'm looking to buy a microscope with phase contrast objectives.
Can I assume these will work well for regular brightfield and darkfield microscopy as well? Are there any disadvantages compared to regular, non phaco objectives?

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daruosha
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Re: Disadvantages to Phase Contrast objectives?

#2 Post by daruosha » Wed Feb 26, 2020 6:11 pm

In "theory", phase plates in the phase contrast objectives, have a negative effect in the image quality, but honestly i cannot tell the difference. They behave just fine in bright field and dark field.
Last edited by daruosha on Wed Feb 26, 2020 7:07 pm, edited 1 time in total.
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Re: Disadvantages to Phase Contrast objectives?

#3 Post by apochronaut » Wed Feb 26, 2020 6:33 pm

Here is the technical reason that phase contrast objectives produce an inferior image when used for BF , or DF for that matter. They will produce an image but it will be subtly inferior to the image produced by an identical objective without the diffraction plates installed. Many oil immersion 100X objectives don't give an image at all in DF.

The diffraction plate in a phase objective, whether it be added to an existing lens element or installed on a separate glass surface creates spherical aberration when used with an illumination source that is non-phase, thus there is a small amount of spherical aberration that occurs with in phase light. There is also a loss of contrast.

If you want excellent imaging, you need two sets of objectives but for average not very critical use, phase objectives will do for a lower quality BF. I wouldn't recommend them for DF

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Re: Disadvantages to Phase Contrast objectives?

#4 Post by daruosha » Wed Feb 26, 2020 9:07 pm

apochronaut wrote:
Wed Feb 26, 2020 6:33 pm
Many oil immersion 100X objectives don't give an image at all in DF.
I never knew this! Note taken. Thank you.
Daruosh.

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Re: Disadvantages to Phase Contrast objectives?

#5 Post by Chris Dee » Thu Feb 27, 2020 12:16 am

I have 2 Zeiss 40x Neofluar objectives, one Ph2 the other vanilla. I've noticed the phase contrast variant performs differently under both oblique and dark field illumination. Its is not severe, but observable when compared. Using bright field illumination I can't see any significant difference between them. HTH.

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Re: Disadvantages to Phase Contrast objectives?

#6 Post by 75RR » Thu Feb 27, 2020 5:32 am

.
Here is a link to an article by David Walker called "Diatom Pleurosigma angulatum - a versatile 'demonstration' subject",

which references the resolution of Phase objectives and includes an internal link to the the subject in "Microscopy Primer", an excellent booklet in itself.

The whole article is well worth reading (as all David's articles are). The phase reference is about halfway down.

http://www.microscopy-uk.org.uk/mag/ind ... -test.html
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Re: Disadvantages to Phase Contrast objectives?

#7 Post by 75RR » Thu Feb 27, 2020 7:25 am

.
The use of Phase objectives in other illumination techniques (usually Brightfield and Oblique) is mostly due to microscopy’s version of diminishing returns:

mainly - is the improvement in resolution from changing the objectives worth the effort of manually unscrewing and replacing each objective on the nosepiece.

If you are merely observing - then probably not, if you are taking photographs then it probably is.

All this can be avoided of course if you have a removable nosepiece – those that do tend to keep Phase objectives

on one and Brightfield on another – swopping them out (it takes just a couple of seconds) as needed.
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Re: Disadvantages to Phase Contrast objectives?

#8 Post by MicroBob » Thu Feb 27, 2020 10:33 am

In some phase setups one phase annulli is used for objectives of different magnification. This will probably lead to differences it the phas effect and the suitability of these objective for bright field work. So probably the single objective has to be evaluated, not a series of objectives.

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Re: Disadvantages to Phase Contrast objectives?

#9 Post by apochronaut » Thu Feb 27, 2020 12:36 pm

Setting up to use a phase contrast system selectively or BF selectively can be accomplished also by fitting a separate derelict stand with one of the systems. Sets of BF objectives for many older microscopes, in fact whole older microscopes are available for peanuts sometimes and sometimes phase systems too.

You don't have to run through the nosepiece usually, so intermixing of phase with BF objectives has been a method of choice for many professional set ups.

In a 5 place nospiece : 10X phase, 20X BF, 40X phase, 60X BF, 100X phase for instance, or other. I've seen a lot of instruments with only 40X phase in a 5 place.

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Re: Disadvantages to Phase Contrast objectives?

#10 Post by daruosha » Thu Feb 27, 2020 1:09 pm

I have setup a separate microscope for phase contrast observation and another one with plan objectives for BF/DF/COL and oblique illumination.

Since i don't have the luxury of a changing nosepieces, this solution works best for me. I aligned the phase rings with each objective and it stays calibrated (I guess each time you replace the nosepiece, you need to realign everything again, not sure).
Last edited by daruosha on Thu Feb 27, 2020 1:39 pm, edited 1 time in total.
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Re: Disadvantages to Phase Contrast objectives?

#11 Post by mrsonchus » Thu Feb 27, 2020 1:31 pm

Hi, I just had a quick look at a couple of images for a rough comparison.
Comparing an Achromat Ph2 40x Olympus infinity objective to an Olympus PlanC N UIS2 'ordinary' BF objective, as image,
WS_phase nosepiece (3).jpg
WS_phase nosepiece (3).jpg (98.68 KiB) Viewed 5805 times
I also keep a phase and BF nosepiece,
WS_phase nosepiece (2).jpg
WS_phase nosepiece (2).jpg (177.22 KiB) Viewed 5805 times
The subject isn't ideal I'm afraid - very red-heavy, a prepared slide of a Sunflower embryo. Taken with a Canon 200D with setting set to 'faithful' which tends to give a sub-optimal (for computer viewing anyway) image. I usually use a user-defined setting with a touch of in-camera sharpening and contrast enhancement to save on PP for 'everyday' images through my BX50. They will suffice though for a quick idea of how close the from-the-camera jpg images are from the phase and non-phase objectives....

I often find that predominantly red and predominantly blue images look quite different with the same settings... I use raw but rarely. I'd agree with the most often aired opinion that the images are, for everyday photomicrocopic images, pretty acceptable. For more critical imaging it seems certain however that the non-phase would be optimal.

A couple of images, this one's from a 40x achromatic Olympus Ph2 objective in BF and Kohler of course - exactly as used for the non-phase version,
Phase 40x used for BF - no in-camera processing or PP of jpg
Phase 40x used for BF - no in-camera processing or PP of jpg
WS_40x phase objective.jpg (83.55 KiB) Viewed 5805 times
This is the same as from a PlanC N UIS2 BF version of the 40x - both objectives are 0.65 n.a.
40x PLanC N BF objective - no in-camera processing or PP of jpg
40x PLanC N BF objective - no in-camera processing or PP of jpg
WS_40x PLANC N objective.jpg (100.7 KiB) Viewed 5805 times
Very slight increase in clarity with the non-phase I'd say - but nothing critical - apologies for a rather 'down & dirty' comparison.
Better images would come from subject with sharper edges but I'm looking over some embryo slides and thought I'd take a couple of comparisons for the forum....

Maybe side-by-side is better,
ContactSheet-001.jpg
ContactSheet-001.jpg (84.5 KiB) Viewed 5805 times
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Re: Disadvantages to Phase Contrast objectives?

#12 Post by 75RR » Thu Feb 27, 2020 1:32 pm

daruosha wrote:
Thu Feb 27, 2020 1:09 pm
I have setup separate microscope for phase contrast observation and another one with plan objectives for BF/DF/COL and oblique illumination.

Since i don't have the luxury of a changing nosepieces, this solution works best for me.
If you don't mind using quality older stands then interchangeable nosepieces are well within reach.

Here is an example of an economical project stand that has decades of life left in it. https://www.ebay.co.uk/itm/Carl-Zeiss-S ... Sweh9eSXHt
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Re: Disadvantages to Phase Contrast objectives?

#13 Post by daruosha » Thu Feb 27, 2020 2:00 pm

mrsonchus, thanks for the comparison. A picture is worth a thousand words. In this case 2 pictures :)

75RR, That's a beauty, but unfortunately i don't the possibility of buying stuff from eBay.
Daruosh.

Goomba
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Re: Disadvantages to Phase Contrast objectives?

#14 Post by Goomba » Thu Feb 27, 2020 2:14 pm

So it seems there is a noticeable difference in contrast and resolution.
But to me it seems like it is not so dramatic as to shell out hundreds of dollars for 2 sets of objectives straight away.

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Re: Disadvantages to Phase Contrast objectives?

#15 Post by mrsonchus » Thu Feb 27, 2020 2:41 pm

ARRGGHHHH!!! Goomba - I just accidentally 'reported' your post - oops - I meant to use the quote button but accidentally used the exclamation-mark (report-post button...) - apologies :oops: :oops:
I'll PP these images also to see just how close they can get to each other. The posted 'faithful' versions from the camera are never optimal I find for photomicrography - but I thought that preset may be good for the comparison. Also - the subject - soft-tissue from the embryo - never shows really sharp edges in slides. A better subject such as perhaps a nice leaf-epidermal edge would have been better maybe?

Back with a couple of PP versions soon....

Here are PS edited versions - virtually the same - close enough to suggest the phase objective is worth having as a stand-in for the non-phase version maybe?
I applied an intensity reduction to the reds of both, a 'smart sharpen' to both, and a slight additional 'unsharp mask' (which unlike the smart-sharpen doesn't look for edges but sharpens 'wholesale' as it were) to the phase objective's image, which seems to have brought the two to a close comparison I think.. Not a great test but better than nothing for a basic idea.


The phase objective...
Edited phase objective version, improved clarity & contrast...
Edited phase objective version, improved clarity & contrast...
PS edited 40x phase objective.jpg (150.05 KiB) Viewed 5749 times

and the non-phase version
Edited non-phase objective...
Edited non-phase objective...
PS edited 40x PLANC N objective.jpg (142.8 KiB) Viewed 5749 times

Side-by-side,
edited image ContactSheet-001.jpg
edited image ContactSheet-001.jpg (106.55 KiB) Viewed 5747 times
Originals,
ContactSheet-001.jpg
ContactSheet-001.jpg (84.5 KiB) Viewed 5747 times
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Re: Disadvantages to Phase Contrast objectives?

#16 Post by Hobbyst46 » Thu Feb 27, 2020 4:56 pm

@mrsonchus
John B.,
The original non post-processed images display significant difference in the hues and in the planarity. Off-center regions are sharper in the BF objective.
Thanks for posting those !
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Re: Disadvantages to Phase Contrast objectives?

#17 Post by apochronaut » Thu Feb 27, 2020 6:18 pm

There is also a significant difference in the two images right smack dab in the center of the field, courtesy of spherical aberration. Colour correction goes off as well as what looks to be a general softness of the image.

If the phase as a BF objective image were to have been presented as a stand alone photomicrograph, I might have been tempted to comment that there was something wrong with the objective.

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Re: Disadvantages to Phase Contrast objectives?

#18 Post by mrsonchus » Thu Feb 27, 2020 6:57 pm

Hi Apo' and Hobby' - I'd agree - definite differences - perhaps made a tad murky by jpg, but they exist. Good enough though for the purchase of a phase objective whilst waiting for a non-phase (assuming the use of phase is also relevant of course).

What we need is some better images for comparison - these are very red and soft from the start.
John B

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Re: Disadvantages to Phase Contrast objectives?

#19 Post by Rossf » Sat Feb 29, 2020 6:04 am

I’ve noticed the difference to be much greater in older objectives-I’ve got a couple of oldies that are abysmal in BF and perfectly good phase-I think in pre University education the “one objective to rule them all” is a great idea-played with the Leica educational models-phase pretty good,BF pretty good-dark field not great but good enough to show bacteria moving about- lets students pick the less eye straining technique-I find Brightfield a bit headachey unless I’m using Apos with lots and lots of glass-tames the light...

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Re: Disadvantages to Phase Contrast objectives?

#20 Post by Rossf » Sat Feb 29, 2020 6:14 am

Good topic BTW everyone-those 6 shooter Olympus nosepiece fully loaded must have a bit of weight to them!

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