NCG vs CG test on 10x

[NikonUser]

Test of the effect of 'no cover glass/cover glass' on IQ of a
Nikon CF Plan 10x/0.3 160/0.17 Achromat microscope objective.
(i.e., designed to be used with a 0.17 mm thick cover glass).

24 frames @0.010 mm ZS PMax. Setup as similar as I could get.

Top: full frame for each face was 2848 px wide. Each face cropped to 2004 px wide, and then resampled to 500px wide.

Bottom: each face 500px wide crop from original 2848 px face

Left image: no cover glass
Right: with cover glass.

Apart from the colour shift and flare with the +CG, there is little difference in IQ.
+CG is perhaps has a little better resolution. Presumanbly the colour shift and glare could be eliminated with more careful lighting.

My bottom line: this lens works OK without a cover glass.


NU10013 & NU10014

[mgoodm3]

Looks better without the CG.

I see a similar difference when shooting through plastic. You lose contrast first, then resolution.

[NikonUser]

Thanks for your comment.
I am not sure that you can equate plastic with a CG. Microscope CGs are designed to be used with microscope objectives and, conversely, certain microscope objectives are designed to work with a CG.

I think the 'problem' is that the objective is designed for transmitted light and then a CG. I used it with reflective light which most likely resulted in some light bouncing back into the lens from the CG.

[Pau]

Nice test
Resolution seems the same, but contrast is far better whithout CG
In my limited knowledge, 0.25-0.30 NA is just under the limit where the objetives became really sensitive to coverglass thickness. Most 10x objectives are marked "/-" , ie, for use with or without CG
In fact the coverglass is best suited to cover a liquid inmersed sample (water or mountant medium). The use of it in air, like in your test seems a good candidate to induce flare.
I think your tests confirm wery well my previous informal tests looking trhough the microscope.

[rjlittlefield]

Well done test, as always.

To put some numbers on the contrast loss, consider that the cover glass adds two new glass/air surfaces in front of the subject. Together, those two surfaces have about 8% reflectance. That doesn't change the bright areas much, but it can wreak havoc on the darks, considering that a good "black" might be only 1% reflectance off the subject.

--Rik

[mgoodm3]

Sounds like you just need to find a multi-coated cover glass to help separate flare from SA.

[NikonUser]

I believe Pau got it correct. It's that 2nd glass/air interface on the cover glass that has a large effect on IQ.The objective expects just one glass/air interface on the upper (front) surface of the CG and then a consistent refractive index (CG/mounting medium/glass slide).

Fortunately only this 10x and the 4x are designed for CGs, all my others, 20x, 40x, 60x, are M Plan and designed as NCG.

And as pointed out by Rik, the 2nd reflective surface of the CG doesn't help.

[rjlittlefield]

To clarify...

What the objective expects is 0.17 mm of cover glass or equivalent refractive index material, somewhere in the optical path.

Where that material is does not affect the ray paths. So from the standpoint of resolution there is no difference between a) having the cover glass adhered to the subject with a vanishingly thin layer of mountant, b) having the cover glass sitting right next to the subject, or c) having the cover glass stuck on the front of the objective or even at some intermediate position.

From the standpoint of flare, position of the cover glass may or may not matter.

Cover glass is normally used with backlighted subjects and some sort of mountant (maybe just water) that reduces or eliminates reflections from the back surface of the cover glass. In this case reflections from the cover glass don't matter much. Any light reflected from the front of the cover glass will simply be directed backward to either the subject or the illumination source. If it hits the subject, it just becomes a bit of front lighting; if it doesn't, it's irrelevant anyway.

When the subject is front lit, then there is a lot more potential for flare from the cover glass. In this case, it would be great advantage to have some multi-coated cover glass. But good luck finding that. Because reflections don't matter for almost all cover glass applications, there's little or no motivation for vendors to make it. I've never seen it advertised, and on a quick scan, I couldn't find it. ITO-coated cover glass is easy to find, but that's for heating, not to reduce reflections.

I think all this is mainly just academic interest. The bottom line is that low-NA objectives work fine with or without the cover glass, and high-NA objectives that give enough working distance for front illumination will probably be designed for no cover glass anyway.

--Rik