Comparison of resolutions of dry 40X Zeiss 160mm objectives with Pleurosira

[Hobbyst46]

Casual tinkering with a 3-years old diatom slide of Pleurosira laevis (mounted in Pleurax) valve, I found that that diatom can serve to distinguish some effects of NA and illumination on the apparent resolution of dry 40X Zeiss 160mm objectives. Although the puncta spacing (0.72um along radial stria in my diatom sample, shown by SEM) of this diatom is not small.

The compared objectives were Neofluar 40X0.75 ("0.75"), Neofluar 40X0.75 Ph2 (phase contrast; "0.75Ph") and Plan 40X0.65 ("0.65"). In fact, the 0.75 and 0.75Ph were compared under at BF, oblique etc in a past thread on the forum. The present experiment extends the comparison somewhat.

The following images were recorded under the same fixed conditions. The 90um-diameter valve was placed hollow side up. LED light was filtered through a green interference filter and rotatable polarizer on the light port, as well as a polarizer between the nosepiece and head. Polarization was assumed to remove some glare. Kohler illumination was set up and the condenser was then fixed in place. Illumination modes included brightfield, oblique and phase contrast.
Tried to exactly focus on the same plane.
Images were cropped and resized to the same extents. Brightness and contrast were tweaked (slightly increased) for visibility.

[Hobbyst46]

So, the above brightfield images indicates a slightly better resolution of the 0.75 than 0.75Ph, yet both are better than the 0.65. The theoretical difference (by the Abbe formula) between the respective performances of 0.75 and 0.65 is less than ~15%.

Next, oblique illumination improves the resolution for all 40X objectives (as expected):

[Hobbyst46]

Finally, an 0.65 image under phase contrast (Ph2). The 40X0.65 is NOT a phase objective. The Ph2 port is exactly set, not shifted. The Ph2 port of the condenser acts as it an oblique, maybe, though in reverse - puncta appear dark rather than bright as in the previous images.

Edit: similar to the 0.65, the 0.75 (again, NOT phase objective) with Ph2 condenser setting yields an image similar to image no. 8 below.

[einman]

Interesting! So, I have read that phase objectives are slightly disadvantaged verse the non phase version but had never actually compared them. This is rather dramatic to me. Do you think this is representative of all Zeiss phase vs non-phase or just this particular phase objective? I recognize you would need more than one, but worth asking.

[Sure Squintsalot]

Was it fair to fix the condenser in the same position for the 40x/0.65 Plan as that of the phase contrast position?

[imkap]

Great, how do you achieve oblique?

[Hobbyst46]

Great, how do you achieve oblique?

Thanks.
For brightfield, I set the condenser to the "J" position, set Kohler illumination, then close the condenser diaphragm for proper contrast. As usual.
To achieve oblique, I then rotate the turret very slightly anticlockwise (could have done clockwise, it does not matter). Just until a distinct improvement of resolution is achieved.

[Hobbyst46]

Was it fair to fix the condenser in the same position for the 40x/0.65 Plan as that of the phase contrast position?

I think so, since:
The bright spot of the sharp image of the closed field aperture remains in focus AND in the exact center of the FOV, no matter if the turret is at the BF ("J") or phase contrast ("Ph2") position. Kohler illumination is preserved, no matter the turret position.
Second, all three objectives are quite parfocal and parcentered. Namely, if I switch any of those 40X objective , the bright spot shifts from the center by less than 50um.
I do not claim that the phase contrast position is "proper" for a non-phase objective. Just noticed the effect, such that playing with illumination modes might improve an image.

[Hobbyst46]

Do you think this is representative of all Zeiss phase vs non-phase or just this particular phase objective? I recognize you would need more than one, but worth asking.

Thanks.
Regretfully, I do not know. In my assortment of objectives collected at random, there are no other exact examples. The closest to a true example could be a Zeiss Planachromat 25X0.45 vs a Zeiss Winkel 25X0.45 Ph2. Just for fun, might test them. At least, the central portion of the FOV, to eliminate the "Plan" factor. Must find a proper diatom for the 0.45NA though. That would be another funny mission .

[imkap]

Great, how do you achieve oblique?

Thanks.
For brightfield, I set the condenser to the "J" position, set Kohler illumination, then close the condenser diaphragm for proper contrast. As usual.
To achieve oblique, I then rotate the turret very slightly anticlockwise (could have done clockwise, it does not matter). Just until a distinct improvement of resolution is achieved.

Great, thanks. I'll remember this when I use the GFL next time