Does phase contrast make sense for its price?

[osterport]

Hi, all

I have a 40x phase contrast objective from Leichter, and I tried quite many times, but the highest resolution picture I got ( no photoshoping ) is like this:

1. There is always a halo around each Yeast cell, which makes the yeast look blur.
2. Due to the fact that the phase contrast stop creates a dark area in each yeast cell, the outer ring of the cell does not show any details, just a bright ring.
3. Phase contrast objective is more expensive than normal ones.

Question: have anyone ever have good pictures with 40x phase contrast? Better take a picture of yeast, very easy to get.

[osterport]

It is not very easy to see the little dots inside the cell actively moving ( pls watch in 1080p )

https://youtu.be/r57YZkvB04A?si=U2B-u4NyPISNxBMT

But if I use a normal 40x motic PLAN objective in darkfield, you can easily see the dots are swimming in the yeast cell.( pls watch in 1080p )

https://youtu.be/6vUnVIsOasY

[PeteM]

Many subjects, such as most pond critters, are somewhat transparent bags of water with semi-transparent features inside. Phase is a good way of adding contrast in those cases and where you don't want to kill and stain live cells.

You do get the halos - and if your main goal is to make stunning photos or videos, then other contrast methods make sense.

Darkfield and oblique can work for some specimens as you've found - though often not with a good look inside. Nikon's apodized phase has a bit less of a halo. Hoffman modulation contrast is an option. DIC can provide a stunning sense of 3D and also have a sectioning effect that tends to give sharp images in single shots and even sharper ones with focus stacking -- but at an even heftier price.

As a very rough generalization, if you want to identify different somewhat transparent cells - phase is probably worth the incremental cost. If you want to make aesthetically pleasing images of the micro world - it may not be worth the incremental cost.

[osterport]

Thanks for the reply. Have you seen anyone with good phase contrast pictures? I took a look at Nikon website, looks quite ok.

https://www.microscopyu.com/techniques/ ... e-contrast

Has anyone here ever used such objective?

[apochronaut]

There really isn't such a thing as just phase contrast but that is what it has been dumbed down to when it comes to new budget microscopes. It's just another example of you get what you pay for.
When you buy a phase contrast kit, or purchase an off the shelf phase contrast microscope, you are getting a very basically designed medium contrast dark phase system but phase is much more than that.
The structure of the phase annulus in an objective can be accomplished by using the most rudimentary of materials and techniques. One commercially made type uses essentially soot, so getting a phase image isn't that hard but refining the image for various contrast levels and additionally a phase inversion takes a high degree of optical engineering skill. Only a few companies have embraced the manufacture of sophisticated phase systems that reduce halo, shading off and define inclusions well but the Chinese systems aren't very good at that. Fortunately , all of that work was already done when Mao was right in the middle of his long march, so a sharply thinking buyer who is interested in a good phase system, need not pay the earth for one. Excellent phase systems were available from the 40's for the next 50 years and are reasonably represented on the second hand market quite cheaply. For about 40 years AO's and Reichert's phase systems blew all others out the door, so it isn't surprising that particularly AO phase systems are well represented in the second hand market because 10s of thousands were sold in North America.One of the most revolutionary discoveries in microbiology was made using AO phase contrast in the 1970's, a discovery that has still not been fully absorbed by the microbiologist community. Ditto for Nikon.

As well, it is difficult to retrofit a microscope to phase, unless you are dead sure you have the spot on parts and know what you are doing. Alignment in all axes is critical and that includes the z axis.

[PeteM]

. . . Has anyone here ever used such objective? . . .

I've used the Nikon CFI (infinity, 60mm parfocal) 10x and 20x apodized objectives, and they do provide less of a halo than others I've used (a dozen or so types). These objectives are really meant for the longer working distance corrections of an inverted infinity microscope - so only the 10x works OK for a normal upright scope - and it wants to be a Nikon Eclipse infinity model.

Zeiss finite phase images, especially with their Plan Neofluar objectives, look very good to my eye - and the scopes and objectives (once you screen for delamination) are comparable in specs and often more affordable on the US used market than Olympus BH2 and Nikon Labophot-Optiphot era phase scopes. Old American Optical scopes, such as the Series 10 can be an even better bargain. The successor Reichert Microstar IV microscopes are sometimes a bargain and have somewhat newer DIN 45mm parfocal objectives.

To simplify - a biologist is likely to find phase contrast revealing. A photographer is likely to find the halos, even in the better systems, a distraction. That said, there has been at least one art photographer who wanted the sort of misty effect.

[Scoper]

How about some representative phase photos of the different systems and types of phase?

[osterport]

How about some representative phase photos of the different systems and types of phase?

that'd be also helpful to figure out some tricks we can make to improve the image quality. I've seen pictures as above, but those are theoretical, do not help us understand the actual results.

[apochronaut]

Yes, pictures help . Those active tutorials provided by those Florida State guys for various mfg., can be useful.

https://www.microscopyu.com/techniques/phase-contrast

[apochronaut]

This is the achilles heel of this forum. It doesn't have stickies, so new members have to be constantly fed with information that is already there but buried in the past. I posted some images years ago but they were done hastily so the image quality isn't A-1. I will see if I can find some.
I don't have a lot of time right now, so I can't really do a series of various images of phase types. I did post this though, which begins the process of categorizing sample types vs. appropriate phase type application. Choosing the right phase type for a given sample is as important as the precision of sample preparation.

viewtopic.php?t=5032

An old post based on an AO series 4.

viewtopic.php?f=6&t=4289&hilit=cicada+head

[osterport]

Yes, pictures help . Those active tutorials provided by those Florida State guys for various mfg., can be useful.

https://www.microscopyu.com/techniques/phase-contrast

Thanks, a lot of information that I need to digest. I'll try something this weekend to see if I can improve the image quality.

Whatever trick is welcome: I see Zeiss has a technique called Varel, but very little information on the internet, have you seen anyone using it?

https://focuspi.com/products-solutions/ ... l-contrast



The varel slide looks like this, I really wonder how it combine phase contrast with relief.


Ideally if varel can work with phase contrast, it'd be perfect.

[apochronaut]

Seems like a phase/ Rheinhart hybrid. Hoffman and the cruciform phase system too.

Phase can be encouraged to provide relief and as well incorporate the value of other techniques for sure. The literature is peppered with thesis papers and post graduate research discovering variations of phase.
I have an ongoing attempt to find all of the phase objectives that were made for the AO series 4., which I now identify as being 28. These are all versions of just 4 achromat objective magnifications: 12 contrast levels of 3 types of phase but not each version was made for each magnification.
In doing so, I ended up with 2 Dark High objectives which I have not found to be catalogued, although they may have been briefly. One of them, the 43X .66 provides very mediocre imaging with excessive contrast until the diaphragm is moved into an oblique condition, where with sufficient precision of adjustment and illumination control, I can obtain a version of DF but with phase attributes, such as highly defined borders and resolved inclusions. It shows a side of phase contrast that may have been little explored. This may have been a prototype objective because I find no reference to dark high contrast objectives anywhere and it's required technique is contradictory to the technique employed with the other objectives in it's family. The objective dates from the mid. 50's.1
Similarly , I have a 40X .70 planfluor infinity corrected Reichert objective with a phase annulus placed lower in the lens stack than the back focal plane. It is also, small and thin walled, completely different than the phase annulus in my similar infinity 40X planapo. It seems to be an objective from one of the Poly microscopes and normally they and the Univar are compatible with the Microstar IV-Diastar phase diaphragm too but this one isn't and not on a Univar either. However, using the 100X diaphragm shifted obliquely this about c.1990 objective gives a very similar DF/phase hybrid image to that of the above 50's objective, just with a higher degree of correction. In both cases there is DF, phase, and some relief.
Oblique/DF/phase hybridization was for sure explored in the past but not a lot was done commercially with it. It was hard enough getting an acceptance of phase in the early days. It is good to see a resurgence of interest.

[Sabatini]

Apocronaut. it would be very instructive and a great opportunity for all to be able to observe images of that unique phase contrast objetive you are describing .
thanks you and merry Christmas for everyone!!!

[Macro_Cosmos]

Whatever trick is welcome: I see Zeiss has a technique called Varel, but very little information on the internet, have you seen anyone using it?

This is something you can absolutely DIY. Get an abbe condenser which allows inserts to be placed at the back focal plane of the objective, and make yourself a slider like this.

I have been able to get this so called Varel contrast thing with my Heine condenser by simply covering up portions. The Heine is not cheap, so a DIY job is preferred.

[osterport]

Whatever trick is welcome: I see Zeiss has a technique called Varel, but very little information on the internet, have you seen anyone using it?

This is something you can absolutely DIY. Get an abbe condenser which allows inserts to be placed at the back focal plane of the objective, and make yourself a slider like this.

I have been able to get this so called Varel contrast thing with my Heine condenser by simply covering up portions. The Heine is not cheap, so a DIY job is preferred.

Thanks. I'm a bit confused: Get an abbe condenser which allows inserts to be placed at the back focal plane of the objective?
My understanding is, I need to DYI a slide like the picture above, and also need to insert a varel ring at the back focal plane of the objective, right? The varel ring is kind of high absorption ring to be inserted into the objective.

By the way, can you share some pictures of the Varel contrast? That'd be interesting for us.

[osterport]

I did a test to compare brightfield, phase contrast, and oblique field. Obviously Oblique field gives much more details, phase contrast only brings some dark area where we do not see anything. ( is it Shade-off artifact?

[osterport]

If we adjust the curve of oblique field image in PS, it looks much better.

[zzffnn]

You can also make DIY circular oblique light (using different circular mask sizes) and cover up 1/3 to 2/3 of the light circle. That would produce similar effect as what Macro_Cosmos does with his Heine condenser.

[osterport]

You can also make DIY circular oblique light (using different circular mask sizes) and cover up 1/3 to 2/3 of the light circle. That would produce similar effect as what Macro_Cosmos does with his Heine condenser.

I'll try it. whether it's 1/3 or 2/3, depends on objective's magnification? how much should it be for 40x?

Another question is the position of condenser, should be lifted to the utmost like touching the slide?

[zzffnn]

How much to block away depends on how much contrast you want for different subjects.

I usually have my condenser immersed and touching slide bottom.

[charlie g]

Hi, osterport, and group. Visual observations with Nikon dl phase finite (circa 1980's?) optics: 10X, 20X, 40X, 60X, and 100X-oil objective..are terrific.

My crude image-capture/ camera setup never records what visually I enjoy with dl-phase contrast methods. And the variety of Nikon phase condenser stops ( ph1, ph2, ph3, ph4) give different degrees of: dark field/ phase contrast, when used with objectives not speced as : 'dl phase objective'...4X Plan objective gives ' some sort of blend of DF, and dl phase' , when used with the ph2 phase stop.

I really would like you to keep in mind your image-capture/camera set up, osterport...is it (like my set up) a so-so mounted : Canon rebel xt dslr camera ( perhaps with projection lens adapter) ?

I say your camera setup really determines your phase image captures to share, to print, to publish. But visually..wow the variety of bacterial morphologies, wow the cilia and flagella , numerous internal organelles are
great microscopy with phase optics.

[charlie g]

Desmid, and hydra with dl-phase contrast method.

[Macro_Cosmos]

Thanks. I'm a bit confused: Get an abbe condenser which allows inserts to be placed at the back focal plane of the objective?
My understanding is, I need to DYI a slide like the picture above, and also need to insert a varel ring at the back focal plane of the objective, right? The varel ring is kind of high absorption ring to be inserted into the objective.

By the way, can you share some pictures of the Varel contrast? That'd be interesting for us.

*of the condenser
Anyway, some manufacturers such as Motic offer Abbe condensers with trays that allow stuff to be plugged in.

I have no examples, I do not find it to be particularly useful for what I do but that was several years ago. I could rig up something over the next few days if I get bored.

[osterport]

Hi, osterport, and group. Visual observations with Nikon dl phase finite (circa 1980's?) optics: 10X, 20X, 40X, 60X, and 100X-oil objective..are terrific.

My crude image-capture/ camera setup never records what visually I enjoy with dl-phase contrast methods. And the variety of Nikon phase condenser stops ( ph1, ph2, ph3, ph4) give different degrees of: dark field/ phase contrast, when used with objectives not speced as : 'dl phase objective'...4X Plan objective gives ' some sort of blend of DF, and dl phase' , when used with the ph2 phase stop.

I really would like you to keep in mind your image-capture/camera set up, osterport...is it (like my set up) a so-so mounted : Canon rebel xt dslr camera ( perhaps with projection lens adapter) ?

I say your camera setup really determines your phase image captures to share, to print, to publish. But visually..wow the variety of bacterial morphologies, wow the cilia and flagella , numerous internal organelles are
great microscopy with phase optics.

Thanks for sharing! I did not use DLSR to record the image, it's just an industrial camera with measuring software. I agree that camera can not record what eyes can see, the 3D effect of stereo image is incomparable.

But still I love to see more details, get the maximum out of the system: like the small dots in yeast, which almost get to the limitation of objective and camera.

By the way, with your 60x or 100x phase objective, do you have pictures of yeast? It's quite easy to take a photo. I want to learn the performance of DL-phase contrast.

[apochronaut]

Unfortunately osterport, your phase contrast contribution above isn't the best and somewhat atypical of what phase is capable of. Some simple phase systems tend to exacerbate the defects associated with achromats, thus using a green filter and photographing in black and white has been employed to overcome that.
It is important to have as thin a sample as possible with phase. The details can be surprising.

[Chas]

My 40x images of yeast are not any better (slightly worse in fact ) however the Cooke Troughton & Simms phase kit I used is not new (cough). The 95x 1.3 does rather better at showing interior detail.

If you look at DIC images of yeast they are maybe the best you could dream for:

https://www.youtube.com/watch?v=PTGYLYru2Sw

To get a sense of what you are looking at, here is a nice fracture of yeast:

https://phys.org/news/2015-07-yeast-key ... ision.html

Do many of the particles really stick out of the cell ??? Nope

It is striking how effective the oblique looks on yeast, but I guess that is because some interior features are refractive oil droplets.

Black (and cream) era phase contrast kits can only be described as 'cheap as chips' (when the ebay fairies are looking kindly upon you) and these make a lot of sense.

[osterport]

Unfortunately osterport, your phase contrast contribution above isn't the best and somewhat atypical of what phase is capable of. Some simple phase systems tend to exacerbate the defects associated with achromats, thus using a green filter and photographing in black and white has been employed to overcome that.
It is important to have as thin a sample as possible with phase. The details can be surprising.

I have a green filter, had tried it some time ago with my old motic 40x NA 0.65 finite phase objective. I found the depth of view is bigger than the leichter phase objective 40x 0.75, look better, the low NA objective resolution is lower. Is this a typical one?

[apochronaut]

That certainly shows more detail. I am wondering how thick your sample is? Yes. Higher N.A. reduces the depth of field but increases the resolution but only if your sample/ coverslip sandwich is within the parameter of thickness that the objective is suited to. The lower N.A. objective can tolerate a thicker more uneven sample, which is obviously why the default 40X objective on an average microscope is .65 and for really simple student scopes, even .55.
Phase contrast is one technique too, that absolutely demands Köhler illumination. It is interesting that DF does not require it at all and PC must have it and no floating coverslips.

[osterport]

That certainly shows more detail. I am wondering how thick your sample is? Yes. Higher N.A. reduces the depth of field but increases the resolution but only if your sample/ coverslip sandwich is within the parameter of thickness that the objective is suited to. The lower N.A. objective can tolerate a thicker more uneven sample, which is obviously why the default 40X objective on an average microscope is .65 and for really simple student scopes, even .55.
Phase contrast is one technique too, that absolutely demands Köhler illumination. It is interesting that DF does not require it at all and PC must have it and no floating coverslips.

From this perspective, I want to increase the depth of field. Is it possible to add an iris at the back of the objective? My Leichert 40x does build-in an Iris, I'm not sure how complicated it is to add one.

[apochronaut]

An iris on a 40X is not uncommon but usually it is on objectives with higher N.A.s. Reducing from .65 will quickly result in a loss of resolution. This brand that you reference , Leichert. I have not seen it and a 40X with an iris is unusual. I know Reichert had a 40X with iris.
It is very difficult to add an iris. Stand alone small irises of various sizes do exist but you would have to find a location in the optical tube to fit it. Putting it in an objective would be tricky,
Prior to the advent of plan optics, when the objective lens stack was all down in the lower part of the barrel, some companies had a two piece barrel. A lens section and a distance piece that threaded into it to position the lens section the correct distance from the nosepiece in order to establish the correct parfocal length. Leitz had an optional iris section that just threaded in to replace that distance piece, turning any so fitted objective into an iris objective. Irises were sometimes used in Pol microscopy as well as in DF.
You can get a similar effect by reducing the N.A. of the condenser with the condenser iris. I often use the condenser iris when I am viewing through PC but usually as a method of increasing contrast with certain subjects. It would follow that depth of field would increase some too.