Moving Sulfur Bacteria

[macnmotion]

I found these quick-moving, long, transparent filamentous strands using a 40X oil objective.i think they are some type of sulfur bacteria, perhaps Beggiatoa or Thiothrix? Can anyone confirm whether these are sulfur bacteria, and if so exactly which type?

Fresh water. FOV 360x200 microns.

[apochronaut]

They look like Beggiatoa to me. If you have DF capability, those sulphur inclusions will irridesce as irregular white spots on the darker bacterial thread.

[macnmotion]

Thanks. I don't have dark field. I've been trying to figure out where I can insert a dark field patch in my inverted Diaphot, maybe inside the phase contrast turret? Not sure. I've looked at 5 or 6 more slides from the sample and haven't run across any more of this bacteria yet but I'll keep looking.

[apochronaut]

There is likely somewhere around there. A lot of the choice of location is based on convenience but the stop works best if it is in focus. The DF occlusion used with a conventional refracting condenser is usually placed at the front focal point. If the phase diaphragms are focused in the condenser then so to would the DF stop, if placed in the same plane. You can get away with some degree of variance in the z plane.
One of the advantages of placing a DF stop in a phase diaphragm port is that you can center the stop against the condenser axis easily.
Have you tried using a larger phase diaphragm with a low magnification objective? I don't know if this is the case for your inverted system, due to the likely L.W.D. design and large phase diapragms but in some upright systems you can usually achieve fairly good DF using the the 100X phase diaphragm for the 20X objective and the 100 or 40X phase diapragm for the 10X and 40X and or 20X diaphragm for lower magnification objectives. Some vertical adjustment of the condenser/diapragm pack may be necessary.

In your video, there are a couple of short segments where the debris causes a blockage of direct rays and a sidelight condition, simulating DF occurs. At those periods, there is a brief negative reversal of the light from transmitted to side reflected I guess, so the dark sulphur inclusions turn white, something along the lines of what happens in DF.

[macnmotion]

Thanks for all the details. I do play with using different phase diaphragms with different power objectives, and get mixed results. There is one combination that does seem to give me something like dark field, but not exactly, more like a blue/gray background . Here are a couple of screen shots showing that.

Screen Shot 2023-01-13 at 22.45.43 copy.jpg (84.94 KiB) Viewed 2506 times

Screen Shot 2023-01-13 at 22.50.46 copy.jpg (58.81 KiB) Viewed 2506 times

[apochronaut]

With low magnification phase objectives and a high magnification phase ring, some of the settings that you would normally use for phase contrast inhibit DF performance. Each microscope has a little different condenser and diaphragms, so there will be variances, particularly with an inverted system where all the distances and sizes of the elements are optimized for l.w.d. and usually a thicker cover.
Your first image seems fairly phase like to me. I don't know what that combination is. The second mimics a condition I can get by using a low magnification BF objective in an upright microscope , for instance a 10X used with a higher magnification phase diaphragm, a 40X for instance . That yields a hot spot in the middle of the field, with a more or less DF corona towards the periphery.
However, using a 10X phase objective and the same 40X phase diaphragm will cause a dark occluded hotspot in the center with a DF corona if the condenser height is not optimized. A slight adjustment of the condenser height extinguishes the dark spot giving a nice dark even DF 100X image.
Therefore with the 4 postion phase carousel and 4 phase objectives the system is capable of 4 phase magnifications and 2 very good dry DF magnifications. You could of course also use the open port for BF, which always entails less than an optimal spherical aberration condition but it is convenient.

[macnmotion]

With low magnification phase objectives and a high magnification phase ring, some of the settings that you would normally use for phase contrast inhibit DF performance. Each microscope has a little different condenser and diaphragms, so there will be variances, particularly with an inverted system where all the distances and sizes of the elements are optimized for l.w.d. and usually a thicker cover.
Your first image seems fairly phase like to me. I don't know what that combination is. The second mimics a condition I can get by using a low magnification BF objective in an upright microscope , for instance a 10X used with a higher magnification phase diaphragm, a 40X for instance . That yields a hot spot in the middle of the field, with a more or less DF corona towards the periphery.
However, using a 10X phase objective and the same 40X phase diaphragm will cause a dark occluded hotspot in the center with a DF corona if the condenser height is not optimized. A slight adjustment of the condenser height extinguishes the dark spot giving a nice dark even DF 100X image.
Therefore with the 4 postion phase carousel and 4 phase objectives the system is capable of 4 phase magnifications and 2 very good dry DF magnifications. You could of course also use the open port for BF, which always entails less than an optimal spherical aberration condition but it is convenient.

Thanks. This microscope was given to me just a few months ago (my first microscope), and I've found woefully little about it online (it's an inverted Nikon Diaphot circa 1985 with Phase Contrast). The phase contrast turret has one open slot (PL) which I use for brightfield, and 3 phase diaphrams (P1, P2, P3). It came with 3 objectives: 10x, 40x and 40x oil. Typically when I film I rotate the phase diaphram turret somewhere in between the empty slot and either the 1st or 3rd diaphragm, in order to optimize my contrast and overall view depending on which objective I'm using and what organism I'm viewing. Other than "playing" with the phase diaphragm turret like that, I don't really understand the optics behind what I'm doing. I rarely (if ever) use the full P1 and P2 diaphragms, they give me some really whacky images. I've adjusted the height of the condensor per the user manual for the microscope, I didn't know I could move it up and down to affect phase views (e.g. using the 10x objective and adjusting condensor height for even DF). The two sample images above were both taken using the full P3 phase diaphragm, the first with the 40x objective and the second with the 10x (as you surmised due to the hot spot in the middle).

Based on that description of what I have at my disposal, can you indicate (or point me to a reference that indicates) which are my best combinations to use for photomicrography?

[Javier]

NIce and relaxing!

Thanks for the additional info.

[apochronaut]

With low magnification phase objectives and a high magnification phase ring, some of the settings that you would normally use for phase contrast inhibit DF performance. Each microscope has a little different condenser and diaphragms, so there will be variances, particularly with an inverted system where all the distances and sizes of the elements are optimized for l.w.d. and usually a thicker cover.
Your first image seems fairly phase like to me. I don't know what that combination is. The second mimics a condition I can get by using a low magnification BF objective in an upright microscope , for instance a 10X used with a higher magnification phase diaphragm, a 40X for instance . That yields a hot spot in the middle of the field, with a more or less DF corona towards the periphery.
However, using a 10X phase objective and the same 40X phase diaphragm will cause a dark occluded hotspot in the center with a DF corona if the condenser height is not optimized. A slight adjustment of the condenser height extinguishes the dark spot giving a nice dark even DF 100X image.
Therefore with the 4 postion phase carousel and 4 phase objectives the system is capable of 4 phase magnifications and 2 very good dry DF magnifications. You could of course also use the open port for BF, which always entails less than an optimal spherical aberration condition but it is convenient.

Thanks. This microscope was given to me just a few months ago (my first microscope), and I've found woefully little about it online (it's an inverted Nikon Diaphot circa 1985 with Phase Contrast). The phase contrast turret has one open slot (PL) which I use for brightfield, and 3 phase diaphrams (P1, P2, P3). It came with 3 objectives: 10x, 40x and 40x oil. Typically when I film I rotate the phase diaphram turret somewhere in between the empty slot and either the 1st or 3rd diaphragm, in order to optimize my contrast and overall view depending on which objective I'm using and what organism I'm viewing. Other than "playing" with the phase diaphragm turret like that, I don't really understand the optics behind what I'm doing. I rarely (if ever) use the full P1 and P2 diaphragms, they give me some really whacky images. I've adjusted the height of the condensor per the user manual for the microscope, I didn't know I could move it up and down to effect phase views (e.g. using the 10x objective and adjusting condensor height for even DF). The two sample images above were both taken using the full P3 phase diaphragm, the first with the 40x objective and the second with the 10x (as you surmised due to the hot spot in the middle).

Based on that description of what I have at my disposal, can you indicate (or point me to a reference that indicates) which are my best combinations to use for photomicrography?

There are two settings that phase requires in order to obtain the most even background illumination. The first is Köhler illumination., which I would assume any phase microscope must be fitted with. The second is a dry condenser. Condensers used for phase are optimized to focus precisely and flat over the entire field. They can however be oiled in order to provide a higher N.A. with immersion BF objectives when the open port is used.

[GerryR]

There are two settings that phase requires in order to obtain the most even background illumination. The first is Köhler illumination., which I would assume any phase microscope must be fitted with. The second is a dry condenser. Condensers used for phase are optimized to focus precisely and flat over the entire field. They can however be oiled in order to provide a higher N.A. with immersion BF objectives when the open port is used.

Interesting, I just started playing with phase contrast and picked up an AO Fifty and subsequently found that the AO One-fifty has the same system for phase. Neither has an adjustable condenser allowing for Kohler illumination, though the One-fifty had an adjustable condenser option, but no field diaphragm, and was not available with the phase contrast version. The condenser lens is fixed right to the stage with only about .003 clearance from the bottom of the specimen slide. I'm now curious as to how "good" are the phase images from the Fifty and One-fifty, and what differences might be expected due to the lack of Kohler??

[Chas]

what differences might be expected due to the lack of Kohler??

I am curious too. (I have been using using a ~2 inch square video light that has a diffusing surface).

really nice video!

[apochronaut]

The more even the background ilumination the better the phase image is going to be. A diffuser will work o.k. but diffusion absorbs light. It would be hard to get a sufficiently bright even background for immersion phase unless you had a pretty healthy illuminator.
On those student microscopes, AO would have optimized the condenser focus for phase contrast and provided some form of diffusion, in order to keep the instrument cost effective. A kind of fixed Köhler . There would have to be some sacrifice in quality.
On the series 10, which does not have Köhler but a modified form of it, they have installed a lens system in the throat of the condenser which changes the front focal point of the condenser altering the illumination system to Köhler. The series 20 uses the exact same phase system but since the 20 has Köhler, that aux. lens system is removed. I am going to hazard a guess that the student phase scopes might have an aux. lens in the condenser throat too.

Phase is greatly affected by the coherence of the wave front. All other things being equal, an illumination system with Köhler would more than likely provide superior phase imaging when compared to a diffusion system, while diffusion would be more economical.

[GerryR]

.... On the series 10, which does not have Köhler but a modified form of it, they have installed a lens system in the throat of the condenser which changes the front focal point of the condenser altering the illumination system to Köhler. The series 20 uses the exact same phase system but since the 20 has Köhler, that aux. lens system is removed. I am going to hazard a guess that the student phase scopes might have an aux. lens in the condenser throat too.

Phase is greatly affected by the coherence of the wave front. All other things being equal, an illumination system with Köhler would more than likely provide superior phase imaging when compared to a diffusion system, while diffusion would be more economical.

In the series 20 manual, they talk about removing a lens if installing a phase condenser from a series 10 unit. I didn't realize what it was for and didn't realize that the series 10 only has a pseudo Kohler system. Having said that, I'm guessing that only a real professional would be able to tell the difference in an image without a side-by-side comparison.
Thanks for the clarification! (Now I'm unclear as to what differentiates the series 20 Kohler from the series 10 pseudo Kohler. Can you explain?)

[apochronaut]

I still like "modified". Pseudo sounds more undignified, although ever since I learned that Pluto has moons I have thought one of them should be named Pseudo Pluto, since it is thought in many circles that Pluto itself is a pseudo planet.

Anyway, the 10 has so many innovative features that it is easy to forget that it is still a microscope with a horseshoe base that first saw the light of day over 60 years ago. That's SIXTY and you would have to pay over $1000.00 for a modern oriental microscope to obtain it's equal. And you still would not have it's phase contrast or planapo capability.
Given it's horseshoe base design they had to cram an entire illumination system between it's legs, not to mention only a few years later, a halogen one as well as it's power supply. In the basic version with a mirror illuminator, the head is turned towards the arm and used like any older horseshoe stand. It is just grey and a bit chunky.

There were a few illuminators designed as free standing units that just kind of plugged in between the legs courtesy of two screws. With the original high end 18 watt incandescent and later 20 watt halogen illuminated models the filament to 45° mirror distance is tiny, so there is no room for the field diaphragm back there adjacent to the collector lens. It was placed forward of the mirror at the illuminator window. It still does it's job well and functions pretty much as a Köhler system, just with the pieces rearranged. Everything is very short and tight in the optical train of the 10's illumination system so the front focal plane of the condensers it used also must conform to it's version of the Köhler principle, with a short focus to a very close field diaphragm. The original condensers #1088 abbe and #1084 abbe aspheric are critically tuned to that task. There must have been another higher N.A. condenser as well, probably a modified focus C235 1.3 achromat aplanat from the series 4 because once on ebay I saw an old 80X 1.3 infinity corrected apochromat dating from the early 60's.

It seems that they conceptualized the series 10 phasestar and H20 phasestar at the same time, rather they conceptualized the phase system at the same time as the series 20 because both came out about the same time. The series 20 only ever had one condenser catalogued for general use , the 1088 which seems odd because they had the short focus #1084 abbe aspheric for the series 10, yet the series 20 was the more research oriented microscope and one would think needed a better condenser than an abbe for some applications. Well in introducing the #1242 phase condenser, they had it : a .90 achromat aplanat which could achieve a higher N.A. oiled plus shortly thereafter the #2110 1.30 N.A. achromat aplanat, which also fits into the phase housing. The latter is very similar in design to the later cat. #1973 1.4 achromat aplanat which they made for the series 400 microscopes.
In order for the 1242 to be used in the series 10 however, it needs the auxillary lens installed. I haven't tried the # 2110 on a series 10. Perhaps it works with the doublet installed too.

[GerryR]

So, basically it is the location of the field diaphragm in reference to the collector lens that determines true Kohler from the "modified" Kohler system. Also, I assume that it is at the higher magnifications where the differences really show up.

[apochronaut]

Not necessariily. Since Köhler is a method of homogenizing the illumination over the field that is seen by the objective, and a low power objective requires a broader field coverage by the illuminator, the lower magnifications could suffer badly from an uneven illumination system., also reducing contrast. The high power objectives require high N.A. and freedom from spherical aberration, which the condenser controls but within that more narrow field a nice even illumination and high contrast is required also.