Testate Amoeba - 2nd image & Question

[NikonUser]

Similar to Charles' Difflugia
HERE
but this is in the genus Centropyxis
Olympus BHS, 20x D Plan Achromat, 2.5 NFK photoeyepiece; flash; ZS PMax.

Width top to bottom, excluding 'spines', 0.22 mm

Shell is very dense and reaches a high region in that very fuzzy region; I suspect there is some protoplasm in this area.

NUM10007
Testate amoeba, on slide in water under cover glass.
20x Oly D Plan with top lighting from a single flash from the side.
Question: would I get a better image with a Oly M Plan objective. I believe M Plans are designed for no cover glass but would the image be sharper with an M Plan and such a specimen under a cover glass?


NU10009

[NikonUser]

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[Charles Krebs]

Question: would I get a better image with a Oly M Plan objective. I believe M Plans are designed for no cover glass but would the image be sharper with an M Plan and such a specimen under a cover glass?

I don't see any reason to think it would be better. If anything, (although at NA 0.40, coverslip should not make a big difference) it might be reasonable to expect a little less. Also, you would need to go back (in time) a little. The Olympus M Plans went to "infinity" type well before the biological objectives did. So you would need an earlier version objective, and they were to be used with an older photo-eyepiece series. (Although I don't know how different they really are from the NFKs).

[NikonUser]

Thanks Charles.
I naively thought that 'regular' objectives expected transmitted light and the M-types expected reflected light.
Thinking about it now, I can't see how an objective could distinguish between a reflected photon and a transmitted one.

[Charles Krebs]

NU,

Well, your thinking was not entirely off base. M type objectives are "expected" to be used with vertical illuminators, where the light comes to the subject through the objective. As such, they are likely to be designed with coatings and lens surface shapes that are intended to keep the reflection of that internal "epi" light to a minimum. But if the light on the subject is supplied "externally" I doubt it makes any difference.

There is one thing you will find as you begin to use the 20X and 40X Plan Apos. The NA's of these objectives means that cover slip thickness, and the "depth" of a subject below the cover slip become more important. The 40X has a correction collar, the 20X does not. You'll come across subjects that are simply too far beneath the surface of the cover slip to get a really sharp image due to spherical aberration. You'll also come across thick subjects (like a testate amoeba) where the portion that is in contact, or very close, to the cover slip are rendered very sharply, but as you run the focus down to the "deeper" section of the subject you can actually notice the fall-off in resolution.

While this is a bigger problem with high NA objectives, it is still an issue to be considered with objectives like your 20/0.40. It tricky with subject like these amoeba tests... you want minimum distance between cover slip and subject, but the subject is fragile and easily crushed by the weight of a coverslip (and even if you have just the right amount of water in the mount, the coverslip will slowly "drop" as the water evaporates from the wet mount).