Do we need Phase Contrast?

Write test posts here. This is the place to experiment with the forum. I may delete the contents from time to time.
Message
Author
komsan.dbg
Posts: 8
Joined: Sat Jun 27, 2020 3:23 pm

Do we need Phase Contrast?

#1 Post by komsan.dbg » Sun Jul 19, 2020 1:32 pm

Hi Microbe Hunters,

1) What is the specimen needed to dye in order to see it in Bright Field?

2) Can we use Phase Contrast to see all of these specimen instead, without dyeing them?

Komsan

Greg Howald
Posts: 1183
Joined: Tue Oct 20, 2020 6:44 am

Re: Do we need Phase Contrast?

#2 Post by Greg Howald » Sat Nov 07, 2020 6:12 pm

The purpose of phase contrast is to observe specimens that are transparent or nearly so. It is a valuable tool, especially if you want to see the specimen in its natural state or while it is living.
Many dyes will damage, kill, or destroy such delicate specimens. Yep. Need phase contrast. Sure do. I don't have it right now. I gave my phase contrast equipment to my brother because it fits his scope. The equipment I need is for my new scope. Cost a little out of range right now.
Greg

PeteM
Posts: 2963
Joined: Tue Dec 06, 2016 6:22 am
Location: N. California

Re: Do we need Phase Contrast?

#3 Post by PeteM » Sat Nov 07, 2020 6:15 pm

Simple polarization, darkfield, and oblique are other and usually less expensive ways of adding contrast.

Phase contrast is excellent for relatively transparent cells (DIC even more so), but typically adds hundreds to the cost of a microscope. DIC even more.

User avatar
mrsonchus
Posts: 4175
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Do we need Phase Contrast?

#4 Post by mrsonchus » Sat Nov 07, 2020 9:08 pm

Phase, very handy sometimes for showing details, especially intra-cellular, of as said unprepared virtually transparent subjects.
Here's a quick example of 'something' from some birdbath water in BF and phase...

Brightfield,
Image

Phase contrast,
Image
John B

apochronaut
Posts: 6233
Joined: Fri May 15, 2015 12:15 am

Re: Do we need Phase Contrast?

#5 Post by apochronaut » Sun Nov 08, 2020 10:17 pm

Excellent comparison mrsonchus. Was there a specific designation to that phase type?

Just to clarify for the thread in general.

Phase Contrast, exists in various types and has been manufactured in most of those forms by one or more companies since about 1946 . Certain microscope systems made by Reichert Austria, American Optical, Olympus, Nikon and PZO ,( maybe Lomo too) offer fairly diverse possibilities in the assembly of a more complete, phase contrast system, broadening it's usefulness.
Phase contrast microscopy has designations of type and contrast level, sometimes abreviatted to a code represented by the first letter of the word describing the phase effect. Dark, Bright, B-Minus,Anoptral,Positive,Negative are terms all associated with phase types and each type is then usually modified by the degree of contrast applied to the type, represented by suffixes such as low, medium, high, l,m,h, or combinations of.
In the 1940's American Optical attempted to coordinate the terminologies associated with phase and suggested a set of universal terminologies. At the time AO was more involved in research into phase contrast than any other company and had already developed working systems of many types. Most other companies except possibly F. Koristka had not advanced very far and the idea of a universal language of phase terminologies was not met with much enthusiasm, since a lot of the systems were as yet non-existent or quite spartan. Later, when Nikon got involved with phase, about 10 years later, they did adopt some of the AO terms loosely abbreviated and subsequently, Olympus did too. They still use them today.
As a result, reading a phase type on the barrel of an objective requires some interpretation and the various phase types and contrast levels provide better or worse imaging for various samples.
If a system is just called generic " phase contrast" or just has ph on the barrel it is assumed to be dark phase and of a medium contrast.

I don't agree that phase contrast is necessarily expensive. Certain used microscope models are relatively expensive on the used market and their accesories too, due to their popularization. For those systems then, phase is probably expensive . However, if you veer off the beaten path and look for various older models of phase microscopes there can be some really good values there. Phase is generally, inconsistent with Bright Field in that you should have separate sets of BF objectives and phase objectives. Some make do with buying a phase microscope and then doing double duty with the phase objectives for BF too. The results are only passable, with lowered contrast and resolution in BF. Most users end up searching out a proper BF objective set.
If you have a BF set already, on a microscope you like and you want to get into phase as well, it makes more sense to look for a complete phase microscope of any brand, than fuss around looking for a phase condenser and set of objectives for your microscope. That's where you pay through the nose likely, or even get fleeced. Sometimes there are good buys in phase accesoties to be found if you stay away from the promoted models but usually, a phase set will cost as much or more than an entire microscope.
I routinely see phase microscopes from various manufacturers for as low as 200.00 or lower, complete. I bought one recently for 65.00, missing one objective out of 4, with a built in bertrand lens. The thing about phase is, that if you have been using BF only, and I don't care what microscope it is, Nikon, Olympus, Zeiss, blah blah blah, then adding the most modest properly functioning phase microscope to your stable will blow you away. You will see organelles previously invisible, you will see cilia previously invisible, you will see whole organisms previously invisible and your depth of field will increase too. Unlike DF with which in order to achieve high resolution you need both an oil immersion paraboloid or cardioid condenser, a high resolution N.A. dampened objective and typically a 100 watt illuminator, high resolution phase can be achieved with any high resolution phase condenser and objective using any standard low wattage illuminator.
Also buying into an "older state of the art system for it's time " phase system , allows the opportunity to acquire a greater diversity of phase types at a fairly low cost. Different types of phase have better representation in older microscopes than newer ones, because most companies are selling, just phase contrast, now.

User avatar
mrsonchus
Posts: 4175
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Do we need Phase Contrast?

#6 Post by mrsonchus » Sun Nov 08, 2020 11:15 pm

Hi Phil, no the phase of my BX50 has no descriptors beyond the usual (generic?) Ph on the barrel. I have the Olympus phase Abbe condenser that is able (after a fashion) to give basic darkfield, and rather acceptable BF to 1.25 n.a. I use the 10x and 40x (both simple achromats at this time), the 10x being recently upgraded to the flatter-topped plan version. I recently found also an acromat 100x oil phase to increase the set's range - but am right now searching for a nice (or any to be honest!) 20x Ph to bridge the gap between the 10x and 40x.
As you mention, I have a seperate dedicated (in terms of objectives on it not it's build) phase nosepiece for the slide-in Olympus dovetail carrier of the BX50 - just as I did with my previous Leitz Orthoplan. Together with the Olympus IF550 the system, although basic, gives me some very nice results, particularly, as you also mention, with organelles - not only their presence and identity but very often their intracellular position, which is most often of relevance also.

A 40x phase and non-phase objective - the green letters are for phase,
Image


Example, pollen-grain nuclei, seperation of nucleus to peripheral position in phase,
Image

The virtually-invisible (in the context of my resin-mounted sections - the very lightly-stained of which I will apply phase to as well as BF) does indeed leap out of nowhere - an experience that in my experience never loses it's power to amaze and delight as a revelation not just technical. The phase (and in general everything really) of the BX50 far outperforms that of it's predecessor but given their historical differences that is likely to be expected I think.

The use of the IF550 with of course monochromatic photomicrography is in my experience, especially with pollen-grain ornamentation and chlorophyll starch-grain inclusions, very nice indeed - giving superb detail and elucidation of tiny characters.... Once aligned the phase rings simply stay put in the revolving condenser-fitting - just as they did with the Leitz version. The phase telescope has many other uses though and I'd advise the purchase of one, together with a 'green' filter, as a definite augmentation with phase.

So in answer to "do we need phase contrast?" I'd say not need perhaps, but have many reasons to desire!
John B

apochronaut
Posts: 6233
Joined: Fri May 15, 2015 12:15 am

Re: Do we need Phase Contrast?

#7 Post by apochronaut » Sun Nov 08, 2020 11:51 pm

I think there are some inexpensive negative phase out there for Olympus systems. I will look into the possibility and p.m. you . Maybe there is something there for you. Those would be bright medium in your system, as opposed to your current system , which are most likely dark medium.
I really like your assessment and useage of phase contrast.

It has become indispensible to me ...in fact my go to system. With the inversions or extensions in the systems, bright, dark, B-Minus, low, medium, high it is sometimes hard to find a use for BF, although every now and then I check in( easy to do with a phase condenser, which is dry and has an open port, with an exceptionally well corrected condenser ), and find something that BF profiles well, so BF becomes a less used but valuable adjunct to phase.
i find that usually that those who pooh pooh phase , just haven't had enough experience around microscopes. Eventually, with time, they come around.

BramHuntingNematodes
Posts: 1536
Joined: Tue Jan 21, 2020 1:29 am
Location: Georgia, USA

Re: Do we need Phase Contrast?

#8 Post by BramHuntingNematodes » Mon Nov 09, 2020 2:59 am

apochronaut wrote:
Sun Nov 08, 2020 10:17 pm

Phase contrast microscopy has designations of type and contrast level, sometimes abreviatted to a code represented by the first letter of the word describing the phase effect. Dark, Bright, B-Minus,Anoptral,Positive,Negative are terms all associated with phase types and each type is then usually modified by the degree of contrast applied to the type, represented by suffixes such as low, medium, high, l,m,h, or combinations of.
[...]

Later, when Nikon got involved with phase, about 10 years later, they did adopt some of the AO terms loosely abbreviated and subsequently, Olympus did too. They still use them today.
As a result, reading a phase type on the barrel of an objective requires some interpretation and the various phase types and contrast levels provide better or worse imaging for various samples.
If a system is just called generic " phase contrast" or just has ph on the barrel it is assumed to be dark phase and of a medium contrast.
I'm looking out for some of the old Tiyoda phase varieties as well. I know they had at least three: dark medium, dark low and bright medium-- they sometimes go for very cheap. The B&L set is very nice, but only dark medium I think.

I don't think phase can substitute for dyeing in all cases, but the idea behind both is similar. Dyeing is hard and messy and unpredictable and really just an unalloyed chore so I would say try to substitute wherever practical.
1942 Bausch and Lomb Series T Dynoptic, Custom Illumination

Hobbyst46
Posts: 4275
Joined: Mon Aug 21, 2017 9:02 pm

Re: Do we need Phase Contrast?

#9 Post by Hobbyst46 » Mon Nov 09, 2020 10:53 am

BramHuntingNematodes wrote:
Mon Nov 09, 2020 2:59 am
I don't think phase can substitute for dyeing in all cases, but the idea behind both is similar. Dyeing is hard and messy and unpredictable and really just an unalloyed chore so I would say try to substitute wherever practical.
Although for me phase contrast is the default, dyeing has one advantage, at least, over phase contrast: The color of certain organelles in a stained preparation can serve as identifier, since it results from specific interactions of the dye with the building blocks of the organelle.

apochronaut
Posts: 6233
Joined: Fri May 15, 2015 12:15 am

Re: Do we need Phase Contrast?

#10 Post by apochronaut » Mon Nov 09, 2020 12:19 pm

BramHuntingNematodes wrote:
Mon Nov 09, 2020 2:59 am
apochronaut wrote:
Sun Nov 08, 2020 10:17 pm

Phase contrast microscopy has designations of type and contrast level, sometimes abreviatted to a code represented by the first letter of the word describing the phase effect. Dark, Bright, B-Minus,Anoptral,Positive,Negative are terms all associated with phase types and each type is then usually modified by the degree of contrast applied to the type, represented by suffixes such as low, medium, high, l,m,h, or combinations of.
[...]

Later, when Nikon got involved with phase, about 10 years later, they did adopt some of the AO terms loosely abbreviated and subsequently, Olympus did too. They still use them today.
As a result, reading a phase type on the barrel of an objective requires some interpretation and the various phase types and contrast levels provide better or worse imaging for various samples.
If a system is just called generic " phase contrast" or just has ph on the barrel it is assumed to be dark phase and of a medium contrast.
I'm looking out for some of the old Tiyoda phase varieties as well. I know they had at least three: dark medium, dark low and bright medium-- they sometimes go for very cheap. The B&L set is very nice, but only dark medium I think.

I don't think phase can substitute for dyeing in all cases, but the idea behind both is similar. Dyeing is hard and messy and unpredictable and really just an unalloyed chore so I would say try to substitute wherever practical.
Yes. I forgot about Tiyoda. They had a fairly full set as well. I think there is a condenser on ebay right now in fact.
Regrettably, Bausch & Lomb did not get into phase too deeply. Although just the default dark system seems to be all, the flat field and planachromat versions are first class.

hans
Posts: 977
Joined: Thu May 28, 2020 11:10 pm
Location: Southern California

Re: Do we need Phase Contrast?

#11 Post by hans » Mon Nov 09, 2020 5:35 pm

Some speculation/questions for discussion:

Is there a name for the contrast effect you get on phase objects with the condenser iris reduced a bit smaller than the objective aperture, as often recommended in Koehler setup procedures?

Is there any fundamental difference in what can be detected with that effect vs. phase contrast other than the subtraction of some background light making visual observation easier?

Are there any shapes of phase object that will give contrast in a phase contrast system but theoretically no contrast in bright field with appropriate condenser iris setting?

Assuming unlimited sensor well capacity and SNR, can digital imaging in bright field with background subtraction done in software always give qualitatively similar results as phase contrast where a similar subtraction is done optically?

User avatar
75RR
Posts: 8207
Joined: Sun Oct 12, 2014 2:34 am
Location: Estepona, Spain

Re: Do we need Phase Contrast?

#12 Post by 75RR » Mon Nov 09, 2020 10:03 pm

hans wrote:
Mon Nov 09, 2020 5:35 pm
Some speculation/questions for discussion:

Is there a name for the contrast effect you get on phase objects with the condenser iris reduced a bit smaller than the objective aperture, as often recommended in Koehler setup procedures?
I wonder if what you are describing is COL

http://www.microscopy-uk.org.uk/mag/ind ... jcol2.html
Zeiss Standard WL (somewhat fashion challenged) & Wild M8
Olympus E-P2 (Micro Four Thirds Camera)

apochronaut
Posts: 6233
Joined: Fri May 15, 2015 12:15 am

Re: Do we need Phase Contrast?

#13 Post by apochronaut » Tue Nov 10, 2020 1:48 am

hans wrote:
Mon Nov 09, 2020 5:35 pm
Some speculation/questions for discussion:

Is there a name for the contrast effect you get on phase objects with the condenser iris reduced a bit smaller than the objective aperture, as often recommended in Koehler setup procedures?
I am guessing that you are not referring to this taking place with a phase microscope. What fo you mean by phase objects? As 75RR suggested , I can only think of some sort of oblique.
hans wrote:
Sat Feb 01, 1975 1:28 pm
Is there any fundamental difference in what can be detected with that effect vs. phase contrast other than the subtraction of some background light making visual observation easier?
Phase contrast has a continuum of qualitative effects, not just the commonly used dark medium type so often seen. Depending on what the effect you are referring to is, it could partly mimic a kind of phase effect, especially if it incorporated a degree of relief, as both oblique and phase can do, however when phase does show relief, it is because oblique or COL has been fused with phase, not because phase has the ability to show relief.
hans wrote:
Sat Feb 01, 1975 1:28 pm
Are there any shapes of phase object that will give contrast in a phase contrast system but theoretically no contrast in bright field with appropriate condenser iris setting?
I don't know. Shape is not normally considered. It is the relative refractive indices and the design of the phase plate that are the primary determinants to whether an object or detail is hard to discern in BF and rendered quite visible in phase.

hans wrote:
Sat Feb 01, 1975 1:28 pm
Assuming unlimited sensor well capacity and SNR, can digital imaging in bright field with background subtraction done in software always give qualitatively similar results as phase contrast where a similar subtraction is done optically?
I have seen some digital systems that attempt to mimic phase. I guess there is some potential there. Obviously the snr would need be adjustable in order to replicate existing phase systems. The question I have is whether the sensitivity of an electronic sensor could be such that it would detect and amplify something that is truly invisible in BF, which a precisely chosen phase contrast optical system could do.

hans
Posts: 977
Joined: Thu May 28, 2020 11:10 pm
Location: Southern California

Re: Do we need Phase Contrast?

#14 Post by hans » Tue Nov 10, 2020 3:15 am

75RR wrote:
Mon Nov 09, 2020 10:03 pm
I wonder if what you are describing is COL
apochronaut wrote:
Tue Nov 10, 2020 1:48 am
I am guessing that you are not referring to this taking place with a phase microscope. What fo you mean by phase objects? As 75RR suggested , I can only think of some sort of oblique.
Not sure if "phase object" is maybe an outdated term or not preferred in more technical contexts but it seems to be commonly used in microscopy context, for example on the Olympus and Nikon reference sites:
Unstained specimens that do not absorb light are called phase objects because they slightly alter the phase of the light diffracted by the specimen...
Light waves that are diffracted and shifted in phase by the specimen (termed a phase object)...
In plain bright field, when the illumination NA is restricted by the condenser iris to be somewhat less than the objective NA, there is a noticeable (although nowhere near as pronounced as in phase contrast) increase in visibility of phase objects which are hard to see when the illumination NA is set somewhat higher than the objective NA. The effect I am talking about is symmetric and does not produce a directional shaded-relief effect like various oblique techniques do. It is often recommended to configure illumination like this, figure 5 in the Zeiss tutorial 75RR linked recently, for example. Tutorials I have seen have little explanation, or only mention light scattered internally in the microscope optics. However I don't think internally-scattered light is the most important effect, at least not with modern objectives.

Here is an interesting demonstration, I think: Put a small speck of immersion oil on a slide and a narrow wedge of aluminum foil with the top colored with black marker next to it and look at them with a 10x objective that has good contrast. When I do this there is very little loss of contrast looking at the foil even opening the condenser iris all the way which I think indicates that internally-scattered light is well-controlled by the design of the objective and not a major factor. However there is still a very noticeable increase in visibility of the oil speck as the condenser NA is decreased below the objective NA. It is that contrasting-enhancing effect on the oil speck that I am talking about. And I think that effect, not loss of contrast due to internal scattering, is the main reason why it is often suggested to restrict the illumination NA to somewhat below objective NA.
apochronaut wrote:
Tue Nov 10, 2020 1:48 am
hans wrote:
Sat Feb 01, 1975 1:28 pm
Are there any shapes of phase object that will give contrast in a phase contrast system but theoretically no contrast in bright field with appropriate condenser iris setting?
I don't know. Shape is not normally considered. It is the relative refractive indices and the design of the phase plate that are the primary determinants to whether an object or detail is hard to discern in BF and rendered quite visible in phase.
Shape is probably not the best term, I meant it in a generalized sense, referring to any spatial variation in refractive index in a transparent specimen, either sharp boundaries between two media or a more gradual gradient.

apochronaut
Posts: 6233
Joined: Fri May 15, 2015 12:15 am

Re: Do we need Phase Contrast?

#15 Post by apochronaut » Tue Nov 10, 2020 11:49 am

There has been a gradual shift in how much aperture reduction is required over time, to the point that at least one company announced that their objectives can be used at full aperture. This would indicate that internal scattering at least until fairly recently was considered a hurdle to be overcome.
Last edited by apochronaut on Tue Nov 10, 2020 1:52 pm, edited 1 time in total.

User avatar
mrsonchus
Posts: 4175
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Do we need Phase Contrast?

#16 Post by mrsonchus » Tue Nov 10, 2020 12:36 pm

Indeed Apo' - I find with my high-dry 60x that there's less contrast as expected with a dry 0.9 n.a. but do find that the correction-collar just about reduces this by about 80-90% when well matched to a slide and coverslip - at that n.a. dry they're all quite individual!

With the 20x 0.7 n.a. without collar-correction (also dry) I have as close to perfection as I've seen (and can afford to see!!). Both objectives are UPlanApo.
I'm after and in serious pursuit of a high n.a. 40x dry Apo to complete the upper dry range as it were...

BOTH of the above are actually at their best at full n.a. - 0.90 and 0.70 respectively - I never need to reduce them, as it happens the 40x 0.65 n.a. Plan achromat performs best at it's maximum n.a. - I don't need to reduce the n.a. below the max for any of the objectives luckily...
Last edited by mrsonchus on Tue Nov 10, 2020 5:15 pm, edited 1 time in total.
John B

hans
Posts: 977
Joined: Thu May 28, 2020 11:10 pm
Location: Southern California

Re: Do we need Phase Contrast?

#17 Post by hans » Tue Nov 10, 2020 5:05 pm

I think there are at least two distinct effects related to condenser aperture opening. Considering only at pure phase objects like small oil drops on a slide, do either of you notice the effect I am talking about where there is an increase in visibility (drops darken toward the edges) when the condenser iris is set close to or just below the objective NA? I suspect this is related to moving the limiting aperture stop for illumination from after the specimen (objective) to before the specimen (condenser iris) allowing refractive effects in the specimen to modulate intensity somewhat. I am not sure to what extent this effect depends on the exact design of the microscope.

The other possible effect, often given as the sole reason for reducing condenser NA in tutorials, is light outside the objective NA being scattered undesirably inside the microscope. I agree this does not seem to be an issue with modern objectives.

apochronaut
Posts: 6233
Joined: Fri May 15, 2015 12:15 am

Re: Do we need Phase Contrast?

#18 Post by apochronaut » Wed Nov 11, 2020 2:38 am

what kind of oil? immersion oil? sandwiched on a slide or on the coverslip or? the refractive index of the oil could vary by as much as .1 depending on type


in order to replicate your demonstration, it might be necessary to duplicate it. right now it is unfamiliar territory.

hans
Posts: 977
Joined: Thu May 28, 2020 11:10 pm
Location: Southern California

Re: Do we need Phase Contrast?

#19 Post by hans » Wed Nov 11, 2020 3:22 am

Yeah, Cargille type 300 immersion oil. I used the little plastic dropper bottle I normally use but didn't squeeze out a normal size drop, just dabbed some of the residue already on the nozzle onto a slide. Drops ~1 mm or less but overall size doesn't seem to matter much, slope and curvature of the surface have more effect. No cover glass just looking at the drops directly on the slide. Then having a dark, opaque object near the drops is useful to see the difference in behavior of "phase object" vs. "amplitude object" to reducing illumination NA. I used a narrow wedge of aluminum foil blackened with sharpie marker small enough to not significantly block the total amount of light getting into the objective, less than 10% of the total field, probably. (If the top surface it not fairly black I think stray light reflected from the bottom of the objective way back down to the top surface degrades apparent contrast below what the objective is capable of.)

apochronaut
Posts: 6233
Joined: Fri May 15, 2015 12:15 am

Re: Do we need Phase Contrast?

#20 Post by apochronaut » Wed Nov 11, 2020 7:54 pm

So the oil has the same n or very close to that of the slide it is on. Thus you are making a very thick plano convex lens. If not the former construction then a doublet. The small diameter results in a rather extreme back curvature which would correspond to the curvature of the front lens of a rather strong somewhat high N.A. objective. You may be stopping down the condenser below the N.A. of your object !

hans
Posts: 977
Joined: Thu May 28, 2020 11:10 pm
Location: Southern California

Re: Do we need Phase Contrast?

#21 Post by hans » Wed Nov 11, 2020 8:34 pm

Yeah, basically little oil drop lenses, which seems like a convenient way to get pure "phase objects" to look at while avoiding other possible confounding effects.
apochronaut wrote:
Wed Nov 11, 2020 7:54 pm
You may be stopping down the condenser below the N.A. of your object !
If by this you mean that the lens-like action of the drops is having an effect similar to varying the condenser diaphragm opening, by effectively changing magnification between the condenser front focal plane and objective rear focal plane for those image points, then I think that is similar to what I was speculating above:
hans wrote:
Tue Nov 10, 2020 5:05 pm
I suspect this is related to moving the limiting aperture stop for illumination from after the specimen (objective) to before the specimen (condenser iris) allowing refractive effects in the specimen to modulate intensity somewhat.
I notice this effect in less artificial situations also, I think. For example in the two amoeba videos I posted recently I believe this effect is responsible for the contrast in the hyaline caps, which become much less visible as the condenser NA is increased above the objective NA. (And as mentioned earlier, I don't think the loss of contrast is related to internal scattering in the microscope, since amplitude objects show no significant loss of contrast during a similar increase in condenser NA.)

apochronaut
Posts: 6233
Joined: Fri May 15, 2015 12:15 am

Re: Do we need Phase Contrast?

#22 Post by apochronaut » Thu Nov 12, 2020 12:03 am

Possibly what you are seeing is a version of shading off, a common phenomena in the interior area of a phase object; under phase illumination,though.

hans
Posts: 977
Joined: Thu May 28, 2020 11:10 pm
Location: Southern California

Re: Do we need Phase Contrast?

#23 Post by hans » Thu Nov 12, 2020 7:09 am

Interesting, does seem possible there could be a similar underlying explanation as shading off (and the halos also are related?), which I have not read much about. Will need to think about it more.

BramHuntingNematodes
Posts: 1536
Joined: Tue Jan 21, 2020 1:29 am
Location: Georgia, USA

Re: Do we need Phase Contrast?

#24 Post by BramHuntingNematodes » Thu Nov 12, 2020 3:53 pm

apochronaut wrote:
Mon Nov 09, 2020 12:19 pm

Regrettably, Bausch & Lomb did not get into phase too deeply. Although just the default dark system seems to be all, the flat field and planachromat versions are first class.
I finally found a 20x Planachromat Phase objective. It was tricky as it had a Leica sticker on it, and I think I probably had to pay an extra $25 for that sticker. I will put it next to the 40x Planachromat with the Cambridge sticker. Have you ever seen any of these rebranded as Reichert? It would be nice to complete my set of stickers if so...
1942 Bausch and Lomb Series T Dynoptic, Custom Illumination

apochronaut
Posts: 6233
Joined: Fri May 15, 2015 12:15 am

Re: Do we need Phase Contrast?

#25 Post by apochronaut » Thu Nov 12, 2020 5:52 pm

I don't think so. You may have to make a fraudulent sticker ! Bausch & Lomb, Cambridge and Leica should be the lot for those. Some AO stuff got Reichert , Cambridge and Leica as well. It seems that very little crossed over from Bausch & Lomb to AO/Reichert and visa versa. There is the one stellar case of the 15X U.W.F. eyepieces that got Bausch & Lomb, FJW, Cambridge, Reichert and Leica.

BramHuntingNematodes
Posts: 1536
Joined: Tue Jan 21, 2020 1:29 am
Location: Georgia, USA

Re: Do we need Phase Contrast?

#26 Post by BramHuntingNematodes » Thu Nov 12, 2020 6:05 pm

Mmm, if scarodactyl still has a pair after I win the lottery I may inquire after those.
1942 Bausch and Lomb Series T Dynoptic, Custom Illumination

henryr
Posts: 182
Joined: Sat Aug 29, 2015 12:29 pm

Re: Do we need Phase Contrast?

#27 Post by henryr » Mon Nov 23, 2020 2:41 am

After reading posts in "Do we need Phase Contrast" I read that some types of specimens can't be seen, well or at all, with a bright field microscope which leads me to the following question. Will bright field prepared specimens be viewable on a phase contrast microscope with its phase contrast condenser and objectives?

Thanks,
henryr

BramHuntingNematodes
Posts: 1536
Joined: Tue Jan 21, 2020 1:29 am
Location: Georgia, USA

Re: Do we need Phase Contrast?

#28 Post by BramHuntingNematodes » Mon Nov 23, 2020 4:28 am

Yes, you can look at bright field with a phase setup, sometimes with small reduction in resolution as a result of interference the phase plate introduces. The manufacturer phase objectives do a p. good job of minimizing this loss I tell you a homemade phase plate I have been having some real trouble with on this front but that's probably not germane to your question
1942 Bausch and Lomb Series T Dynoptic, Custom Illumination

BramHuntingNematodes
Posts: 1536
Joined: Tue Jan 21, 2020 1:29 am
Location: Georgia, USA

Re: Do we need Phase Contrast?

#29 Post by BramHuntingNematodes » Mon Nov 23, 2020 4:32 am

Oh also you can often do dark field and circular oblique with a rotary phase condenser and normal bf objectives in addition to regular bf, so that's neat
1942 Bausch and Lomb Series T Dynoptic, Custom Illumination

apochronaut
Posts: 6233
Joined: Fri May 15, 2015 12:15 am

Re: Do we need Phase Contrast?

#30 Post by apochronaut » Mon Nov 23, 2020 1:10 pm

henryr wrote:
Mon Nov 23, 2020 2:41 am
After reading posts in "Do we need Phase Contrast" I read that some types of specimens can't be seen, well or at all, with a bright field microscope which leads me to the following question. Will bright field prepared specimens be viewable on a phase contrast microscope with its phase contrast condenser and objectives?

Thanks,
henryr
Specimens prepared for BF can be prepared in many ways and normally that same preparation is applicable to phase contrast as well. Exceptions might be preparations with blocky or thick sections, such as algae clumps where the innate contrast is high or certain stained subjects, with artificially enhanced contrast.
Phase contrast converts a phase difference into an amplitude difference and therefore an intensity difference between two objects, thus increasing the contrast between them. This means that most samples that have low contrast under BF are perfect for phase.
The two biggest mistakes that take place when using phase are 1) misalignment in the illumination pathway. Even a small misalignment will alter the accuracy of the illumination beam , so that the phase shift is not consistent across the field. This seems to lower the overall contrast/resolution quotient or that is what appears to me to be the case. I haven't investigated the effect fully, yet. 2) misalignment of the diaphragm and phase plate. Even a tiny misalignment has a negative effect on image quality and coupled with 1 above, a considerable effect. Any and all ways of limiting diffraction will benefit phase imaging.
Phase has a unique ability to limit the negative aspects of achromat objectives. Where chroma has an obvious deleterious effect on an image in BF, the same field imaged in well aligned phase is much less affected. Misalignment of the optical path in any way, increases diffraction and chroma. That is why phase condensers are so precisely fitted to a system. You cannot just swap a phase condenser for another type and expect excellent results. Phase condensers can be expected to perform adequately in BF mode, although phase objectives induce spherical aberration into BF and have limited use but standard BF condensers are usually not adequate for fine phase imaging. Even highly corrected achromat/aplanat condensers that were not originally designed to be used in a phase system can provide poorer phase contrast imaging than an abbe condenser originally designed to be used for phase.

Post Reply