AO series 4 phase contrast comparison images.

[apochronaut]

Looking into the illumination specs. for Rod's apochromat equipped series 4, I decided to do some comparison shots of the different phase objectives that I have for the series 4. AO made 24 different phase objectives for the series 2/4. The most in any one magnification, I can come up with is 4 -10x:
Dark Low contrast, Dark Medium contrast, Bright Medium contrast and B-Minus Low contrast. In the 10x magnification, they also made a Bright Low contrast.
I am looking to complete the set of objectives that I have, if anyone has some to let go. I need 11 more and will post a list in another thread.

First set of 4. A Cicada head.

[apochronaut]

second set; Bryophyta.

[rnabholz]

Very interesting set.

I don't see many Bright Phase images out there. I am sure there must be, but are there specific applications where bright phase is considered advantageous?

[zzffnn]

I love this comparison! Thank you for doing this, Apo.

I have only ever seen one or two other B-Minus images eslewhere, and those came from you as well.

My eyes much prefer the Dark M/L and B-Minus, over the Bright M.

With the Bryophyta, my preference is Dark M > L > B-Mius >> Bright M.

With the Cicada head, the better three performed about the same to my eyes, and still much better than Bright M.

I am surprised that the Cicada head did not produce too much halos in any phase objective. I would think it would be too thick for good phase contrast, even at a low 10x NA 0.25.

Could your Bright M objective have some defect?

[apochronaut]

Very interesting set.

I don't see many Bright Phase images out there. I am sure there must be, but are there specific applications where bright phase is considered advantageous?

I guess where the main body of the sample might have a darker matrix , the bright phase would give better contrast to transparent bodies one might be looking for and visa versa for dark phase. I think when AO first marketed phase, around 1948 or so, they seemed to put a lot of emphasis on bright phase but the ultimate value of the technique's various incarnations was market driven. Dark phase turned out to be more generally useful and by the time the series 100 was offered ,only dark( M, I believe) was offered and almost all other companies, just offer dark. Perhaps NIkon , still has a bit of variety...I;m not up on that .
For my part, since I have at least one objective of each of the 7 phase types offered by AO originally, I can see a use for each. The market must have been small for B-MInus, though. It shows inclusions well and differentiates, very lightly pigmented bodies well. It must have had many industrial applications but by the time the series 10 came out, they had whittled the phase objective offerings down to 9 : 4 bright( M) , 4 dark (M) and 1( 100x) B-Minus L. The B-MInus L would be of value in parasitology, maybe blood smears and with some chemical compounds and polymers, fibres etc.

[apochronaut]

I love this comparison! Thank you for doing this, Apo.

I have only ever seen one or two other B-Minus images eslewhere, and those came from you as well.

My eyes much prefer the Dark M/L and B-Minus, over the Bright M.

With the Bryophyta, my preference is Dark M > L > B-Mius >> Bright M.

With the Cicada head, the better three performed about the same to my eyes, and still much better than Bright M.

I am surprised that the Cicada head did not produce too much halos in any phase objective. I would think it would be too thick for good phase contrast, even at a low 10x NA 0.25.

Could your Bright M objective have some defect?

Thanks, Fan.
I don't think so, about the objective.. The 10x bright M seems fairly consistent with what the 43x Bright L and 97x Bright M , are capable of; the other brights, I have I don't have a 20 bright. Kurt, has a bright phase , series 10. perhaps you could weigh in, Kurt?

It's probably the sample(s) but I do like the highlighting of the hairs, against the dark background. I'll check the objective over, in case I smudged it with finger or something.
I remember you had an interest in such a comparison, Fan; very glad it met with your approval. I should do a blood smear with Dark M, Bright M and B-Minus L. all 97x. It would be interesting to see the differences in their ability to highlight inclusions.

[zzffnn]

I should do a blood smear with Dark M, Bright M and B-Minus L. all 97x. It would be interesting to see the differences in their ability to highlight inclusions.

^ That would be great, thank you apo. Looking at an unstained white cells may reveal some difference.

[75RR]

How are each of the Phase types achieved? Is it a combination of annulus size, position and width or is there more to it than that?

[KurtM]

Kurt, has a bright phase , series 10. perhaps you could weigh in, Kurt?

True. But I'm not gonna lie to you ... I'm in the slow lane, and when Fan and Phil are having a technical discussion I get left behind like I was tied to a tree stump. What's the question?

And may I ask why a cicada head is being used as a test subject for phase contrast? Seems like a much better suited choice would be a transparent subject that offered very little contrast in bright field, like an amoeba for instance. Or better yet, a stationary target like a diatom frustule. Which is what most of us are familiar with anyway? I had to Google "bryophyte" to remind myself it means moss. Not really a notoriously transparent subject needing enhanced contrast techniques either?

But I sure know what a Bryazoan is. It's a subject Rod and I really ought to revisit one of these days. But not with phase contrast...

[apochronaut]

How are each of the Phase types achieved? Is it a combination of annulus size, position and width or is there more to it than that?

I 'm pretty sure that the phase ring in bright phase retards the wave a portion and the surround of the phase ring in dark phase retards the wave, causing an inverse effect. The phase rings are the same size and are located similarly, if not in the identical location. I'd have to take the objectives apart and do some pretty precise measuring, in order to determine that with accuracy.
B-Minus has no discernible phase ring and does not diminish the central wave. There are probably some slight wave diminishing coatings that partially slow some part of the wave down so that a low level of phase occurs.

[apochronaut]

Kurt, has a bright phase , series 10. perhaps you could weigh in, Kurt?

True. But I'm not gonna lie to you ... I'm in the slow lane, and when Fan and Phil are having a technical discussion I get left behind like I was tied to a tree stump. What's the question?

And may I ask why a cicada head is being used as a test subject for phase contrast? Seems like a much better suited choice would be a transparent subject that offered very little contrast in bright field, like an amoeba for instance. Or better yet, a stationary target like a diatom frustule. Which is what most of us are familiar with anyway? I had to Google "bryophyte" to remind myself it means moss. Not really a notoriously transparent subject needing enhanced contrast techniques either?

But I sure know what a Bryazoan is. It's a subject Rod and I really ought to revisit one of these days. But not with phase contrast...

I was just scrounging around looking for something big enough that the 4- 10x objectives would resolve something of interest. I did choose a section of the moss that did harbour a diatom but when I photographed a diatom slide, there wasn't much to see. Insect hairs, pores and convolutions on the shell seemed o.k. at the time.... was just interested in a comment about bright phase, from you Kurt.

[zzffnn]

Kurt,

Do you prefer dark phase or bright phase for pond protists?

You are too humble. I am just a cheap guy with small toy funds trying to get the best of my money - I do lots of research, reading and procrastination, during which time I learn by harassing pros like Phil.

[zzffnn]

I think one or both of the following had drawings re bright vs dark vs B minus phase:

https://user.xmission.com/~psneeley/Per ... aseMan.zip

https://user.xmission.com/~psneeley/Per ... aseCat.zip

Basically, difference was achieved by different (objective) phase plate opacity, IIRC.

[einman]

Very nice..definitely see the difference. You even used an insect!! I do believe the first insect slide I have seen you post..and good ones at that.

[KurtM]

Kurt, Do you prefer dark phase or bright phase for pond protists?

The answer is: yes.

Seriously, I feel like a broken record when I say I can never pick any one lighting method as a favorite, since each has its strengths and weaknesses. The winner is he who has the most lighting methods at his disposal. When looking at pond protists, I often start with bright field, go to dark field, then to phase contrast, then try POL (or substitute DIC in place of phase if using my Zeiss WL instead of the AO120).

Plus, not all objectives perform equally. My bright phase objectives aren't the same type as my dark phase objectives.

Plus, how they perform to the human eye versus how they perform to the camera sensor can vary a lot.

Plus, going back to where I started, when viewing the same target bright phase shows some details better than dark phase, but dark phase shows other details better than bright phase.

Now, if you were prepared to purchase a phase contrast system and had the choice of which to get (and all other things being equal), then which would I recommend? The answer would have to be "flip a coin". But one thing's for certain: I'd have to say you couldn't go wrong either way - I'm a huge fan of phase contrast. YMMV, of course.

Incidentally, both my phase contrast equipped Olympus KHC and Nikon Labophot stands have bright phase. I really see it as six of one and a half dozen of the other.

EDIT: Here is a comparison I shot a while back, first bright phase, and last one dark phase. The bright phase image shows better detail; but visually, the dark phase view is on a par with the bright phase view.

[Crater Eddie]

I should do a blood smear with Dark M, Bright M and B-Minus L. all 97x. It would be interesting to see the differences in their ability to highlight inclusions.

Yes, please do. I would love to see that.
CE

[zzffnn]

.

[zzffnn]

EDIT: Here is a comparison I shot a while back, first bright phase, and last one dark phase. The bright phase image shows better detail; but visually, the dark phase view is on a par with the bright phase view.

Thank you, Kurt. Bright phase worked much better there, to my eyes!

Hmm......

Did you take those two phase photos on the same scope, with same amount of light? I wonder what if you give dark phase more light, would that produce more details or just more halos?

[75RR]

EDIT: Here is a comparison I shot a while back, first bright phase, and last one dark phase. The bright phase image shows better detail; but visually, the dark phase view is on a par with the bright phase view.

Clear difference in your images between bright and dark phase - must say that the differences between the original poster's images are quite subtle and seem to lie within the range of normal setup variations.
Did you do much post processing on them?

[apochronaut]

must say that the differences between the original poster's images are quite subtle and seem to lie within the range of normal setup variations.

can you explain how, in normal settup variations, one can get insect hairs that show up black in one picture, to turn white in another?

[75RR]

...can you explain how, in normal settup variations, one can get insect hairs that show up black in one picture, to turn white in another?

Not much between Dark L 10x .25 phase achromat, Dark M 10x .25 phase achromat and B-Minus L 10x .25 phase achromat.
Bright M 10x .25 phase achromat does show some differences (re your reference to change of hair colour for example), though not as marked as KurtM's images show. Hence the question as to the amount of post processing done.

[zzffnn]

Phil,

Would you please find a big and easy diatom, then image it with all 3 phase types? I don't know if the Klaus Kemp test slide I sent you still have those intact or not.

Kurt's bright vs dark phase comparison indicates that diatom may reveal more difference.

It would be interesting to see, especially for the many diatomists here on this forum, including myself, Rod, Kurt, Dale, Charles and exmarine.

[apochronaut]

...can you explain how, in normal settup variations, one can get insect hairs that show up black in one picture, to turn white in another?

Not much between Dark L 10x .25 phase achromat, Dark M 10x .25 phase achromat and B-Minus L 10x .25 phase achromat.
Bright M 10x .25 phase achromat does show some differences (re your reference to change of hair colour for example), though not as marked as KurtM's images show. Hence the question as to the amount of post processing done.

It is probably due to the thickness of the sample, so the phase changes aren't that evident throughout most of the subject. My broadest range of different objectives is in the 10x, although the one extra is just Dark L, which is only subtly lighter than the Dark M, so I chose those and a fairly large subject for the comparison.
If the thinner parts of the cicada's head are focused on, the differences are more evident. The B-MInus L however, does do as advertised by AO....it manages to separate inclusions. If you look at the two lance shaped bands on either side of the central crest of the head, the hairs on the left one are only really revealed well, by the B-Minus L objective. Similarly, the Bright M objective manages to "see" through the rather heavy structure of the same area to reveal details of the understructure that are only hinted at by the Dark phase objectives. So, each has it's uses, depending on the structure and the needs of the microscopist.
Post processing was minimal. A little sharpening and an attempt to even out the background by adjusting the shading a little.

[KurtM]

The two images I posted are really rather poorly suited for comparison purposes. They were taken through two totally different microscopes, at two totally different times, and focused on two different planes, even. I never envisioned comparing them until long after the fact - they were merely dug up out of the archives and placed side by side. If we are to be fair, I must try again with this discussion in mind. Perhaps I will, later on today if I have time, as I'm going out of town tomorrow morning.

This reminds me that dark phase diatom images through the 100x lens tend to look underexposed, and that is due to the high dynamic range of highs and lows. Similar to the difficulties dark field imaging presents, when you keep the brightest areas from blowing out altogether, the dark areas tend to be too dark. This isn't nearly as prevalent with bright phase contrast. Also, this pertains to imaging only, as the human eye encounters no such difficulty.

75, I post process them without mercy. They're cropped, light levels adjusted, sharpened, saturation and luminescence levels toyed with, etc. I may not be very good at it, but that doesn't even slow me down...

[apochronaut]

Here are 3 quickies of some sort of cymbella 43x .66 N.A. They are heavily cropped to isolate the diatom, the background has been evened out some and they have been sharpened.

[KurtM]

That B Minus looks pretty convincing. I want.

Okay, here are a few I just shot of a Klaus Kemp slide labeled simply "Phytoplankton", chosen for its ultra-low contrast specimens. Dark phase through the AO 120, bright phase through the AO 10, both converted to LED. Images are cropped to square, otherwise full size with no other post processing done.

1. Dark phase 40x
2. Same, only adjusted exposure to illustrate how brights get blown out.
3. Bright phase 40x
4. Bright field 40x

I have also heard of dark phase and bright phase referred to as positive phase and negative phase, and these images show why.

[zzffnn]

Very cool stuff, thank you very much, Phil and Kurt.

Overall, so far, my favorite is the B-Minus for protists. Its ability to image inclusion and see through should provide better internal details of protists, thus helping with identification.

I wonder how much better is B-Minus over oblique or brightfield with iris way closed down, for an unstained white cell or any low contrast thin/small subject. I am guessing quite a bit?

Bright phase does seem to have an edge, over dark phase, with diatoms. The halos can be distracting though, sometimes.

[apochronaut]

Thanks for that, Kurt. I find the dramatic difference in the imaging of those forked structures( one about 2:00), very interesting. Makes one wonder what they are mistaking things for? The bright phase makes it look like a crack in some crystalline substance. The BF seems to image the large prominent diatom the best.
FYI; they only made 1 B-Minus objective for the infinity scopes, a 100x 1.25 achromat. B-Minus L. B-Minus M is sometimes too contrasty, stark really. For the 160mm tube length microscopes, they made a complete set of B-Minus low contrast and a 43x and 97x B-MInus medium contrast. Fairly hard to find.

[apochronaut]

I should do a blood smear with Dark M, Bright M and B-Minus L. all 97x. It would be interesting to see the differences in their ability to highlight inclusions.

Yes, please do. I would love to see that.
CE

I took a few quick pictures of a blood smear, using the above objectives, Crater Eddie.
viewtopic.php?f=6&t=4338

[Crater Eddie]

Thanks!
CE