Alga in Brightfield/Phase + Oblique Test

[75RR]

25x, 40µm, Brightfield, Brightfield+Oblique and Phase, Phase+Oblique 8, 6, 6, and 6 image stacks in Photoshop

The idea was to use the same subject in order to provide a valid comparison of the Oblique effect.

Set the White Balance in Phase but did not increase illumination which is why the background is grey rather than the more common blue or blue/grey and the alga is correspondingly darker. Not an effect that works well in this case.

[billbillt]

Not sure what your intent was here, but all of these are utterly outstanding!.....

[75RR]

Thanks billbillt.

I find Oblique quite effective, just showing views with (right) and without (left).

[75RR]

Double post

[jwsmith]

oblique, in both cases, seems to provide greater definition.
I spent several moments looking back and forth....
Contrary to my first impression, brightfield contained ALL the information that phase did.

When comparing "definition" any use of "stacking" infringes upon any conviction of clear comparison.
I have had "good stacks.."..
I have had "bad stacks.."....
They definitely influence final impression of "resolution" and "detail" and "quality."

Nothing is exactly perfect....in observation.....
Unless you have SEM.......SEM looks pretty perfect...
But gee...

Judd

[gekko]

Beautiful images. But I agree with Judd: I would have avoided focus stacking for the purpose of comparing the different lighting techniques-- it sort of muddies the water, I think. Otherwise, beautiful; I especially prefer the BF oblique over the straight BF, but my eyes do not see much difference in the phase images (not that there isn't).

[75RR]

Thanks gekko

But I agree with Judd: I would have avoided focus stacking for the purpose of comparing the different lighting techniques-- it sort of muddies the water, I think.

It does add another variable but assuming the stacks are comparable the difference in technique should still stand out.

... my eyes do not see much difference in the phase images ...

You are right there is not much. If I had increased the illumination I would have been able to play with the Oblique a bit more. My mistake. Still, I have learned to leave Oblique more leeway, especially in Phase.

[vasselle]

Bonjour.
Très belles photos.
Eclairage oblique est un bon moyen pour mettre en évidences les détails.
Cordialement seb

[apochronaut]

I like this comparison, although I am surprised that the phase , suppressed the oblique contrast a little . Perhaps a reverse phase( bright, anoptral or whatever the mfg. calls it) might have worked better with this sample.
Oblique illumination was much used in the past. Most of the older research microscopes had an oblique diaphragm, with crescent shaped leaves available as an add on to the substage. It was really the first, contrast enhancing tool, beyond the straight manipulation of the optic/condenser set.
When phase contrast and D.I.C. arrived on the scene, it became a little forgotten but in combination with phase or alone, it can be very useful and trouble free to set up. Getting it fairly even, as you have done here, is the trick; when skewed too much ,it begins to obscure ,rather than enhance.
Oblique + phase can render a quite accurate impression of topography, whereas D.I.C., although relied on as a better imaging tool, renders a completely artificial impression of topography.
Here's a diatom , that I am unfamiliar with that I took in bright and dark phase with slight oblique illumination.

[75RR]

Thanks apochronaut
I see more detail on the 40X bright phase with slight oblique.
Does bright/dark phase refer to the amount of illumination you gave it or is it a type of illumination?
A rotating stage is the next step in order to be able to alter the angle I can illuminate the subject from.

[apochronaut]

It isn't related to the illumination, rather to the wave retardation of the phase ring. Bright phase retards the waves passing through the annular ring , which has the effect of causing particles with a higher refractive index than the surrounding medium to appear bright. Dark phase retards the waves adjacent to the annular ring , which has the opposite effect. There were lots of different phase options in the early years, particularly from AO , who put a great deal of energy into developing phase as the contrast enhancing technique par excellence but D.I.C. came along just as they were really rolling with it and kind of stole the show. At one time( between the late 40's and 1960) AO had 7 different types of phase contrast systems and by 1965 or so they were down to 2( bright and dark), then shortly after, 1; dark phase. Originally, I think bright phase was the one that held the most promise.
Those diatom pictures aren't the best to illustrate the differences due to the complex structure of the sample. I'll see what else I can come up with, if you don't mind me adding this in to your post thread?

[75RR]

Not at all. Most instructive.

[Crater Eddie]

Nice comparison sequence. They actually make interesting "cross eyed 3D" sets.
CE

[gekko]

apochronaut: superb images. I hope we will see more at some point.

[apochronaut]

Here is a comparison of 3 of the 7 types of phase contrast, originally sold by AO. I have only 12 objectives. If this sounds like a lot , the full complement was Bright high,43x and 97x; Bright med.10,20,43 and 97; Bright Low 10,20,43 and 97 ; Dark med. 10,20,43 and 97 ; Dark low 10,20,43 and 97 ; B-Minus med. 43 and 97 ; B-Minus low 10,20,43 and 97., for a total of 24. The series 4 microscope itself, which was the most complete instrument using these systems, has a quick change nosepiece, so various phase sets could be installed in seconds.
There may be others but I have only seen Nikon and Reichert( under AO's tutelage, probably), offer much more of a range , than just phase contrast. The type most often offered appear to be a dark medium contrast version, by most companies.

B-Minus does not have a an annuli in the objective, rather it has a wave retarding plate, kind of like a mini dark field stop. You can't see it, looking straight through the objective but you can if it is tipped to a bit of an angle. The effect of this type is a kind of enhanced brightfield. Contrastier portions of the image appear with more contrast, the background starker ,more evenly white and very transparent structures, such as cell borders and the like are given sufficient contrast to be visible, when in bright field they are barely so. B-Minus medium has quite a stark contrast, enough so that B-Minus high was not offered, nor was Dark high contrast.

[75RR]

Thanks for the additional info apochronaut.
AO truly went whole hog on Phase.

The type most often offered appear to be a dark medium contrast version, by most companies.

If that includes Zeiss then fortunately dark medium does appear to be the best of the 3 you have shown!
There would appear to be a lot of "old" interesting stuff out there if one has a microscope that can use it.

[apochronaut]

In AO's literature , they pointed out that the different phase systems, had specific applications. Choosing the right contrast system for the application was important. In these pictures , the Dark M contrast does bring forth a crisper definition to the surface features of this fragment of tissue but it has much less resolution of fine details that are within the tissue or on or above it's surface.
When I was focusing the images, I used a dividing bacillus on the tissue surface as a guide. Just getting the division between the two cleaving cells was barely possible and only possible with the Bright M and the B-Minus. If you look at the Dark M image, and locate the two bright yellow spots at the upper left of the tissue( these were actually chlorophyll bearing bodies) and then draw a line between them to the right, to intersect with a line drawn down from the point at the top, there is a little dark diagonal slash at that intersection. It is lined up pretty close to an angle from about 10:30-4:30. That's a dividing bacillus. I could not get the Dark M objective to focus the division. It is somewhat visible on the Bright M image and very visible on the B-Minus L image.
On the upper right, there are two " fingers" that stick up and to the right, near the top , making what looks like a cove or bay between them. The lower of those two " fingers", had some inclusions that are clearly visible as little dots in the B-Minus L photo but are completely unresolved in the pictures taken in Dark M and Bright M.

[75RR]

In AO's literature , they pointed out that the different phase systems, had specific applications.

I see what you mean about the different views each one provides.
Agree the division of the dividing bacillus is visible in B-Minus and Bright M, yet not in Dark M.
I can also see the dots on the lower "finger" in B-Minus though I believe they are also just visible in Dark-M.


One of the things I like about Oblique is that it gives one an additional tool, just varying the light intensity is a little limiting.

[Charles]

Phil,
Speaking of AO Phase variations, I have quite a few myself:
Dark M and Bright M full sets (10X, 20X, 43X and 97X)
Also have:
Dark L in 43X and 97X
Bright L in 97X
Bright H in 97X
Dark H in 97X
B Minus L in 20X (older no ring objective)
B Minus M in 97X

These can also be used on the Model 15 and 35. My first AO phase set I purchased came on a 15.

Also have full sets of two variations in the 'ringed' infinity phase objectives, Bright Phase and Dark Phase (10X, 20X, 45X, 100X) and a few sets in the newer modern Infinity sets as well as some for the inverted scope.

I've only used the regular Bright and Dark phase sets but now you have piqued my interest and am intrigued in seeing what the others look like.

Here are some of the sets and extras. The Infinity sets are on the left:

AO Phase Sets

AO Phase Sets.jpg (79.05 KiB) Viewed 6417 times