Greetings all.
The attached image of a (cracked) T. favus diatom is a focus-stack of 70 images captured under bright field illumination on an Olympus BX61. I used a UPlanFL 40x/0.75 dry objective and a Sony A7riv camera. Focus-bracketing and image capture were automated with a WeMacro Micromate attached to the focus knob. I inverted the image to negative in post to get the darkfield look.
Those of you familiar with the output of a typical 40/0.75 dry objective (capturing diatoms) may notice this image has significantly better resolution than usual. That's because...
The camera was converted for full-spectrum and I used 365nm UV light. This shorter wavelength improves the maximum 0.45µm resolution of the objective (in green light) to 0.30µm. Also...
The objective is not corrected for UV so I had to move it closer to the specimen to get focus. This lets the lens collect more, steeper-angle (higher NA) rays from the specimen, effectively increasing its NA to 0.85 (or more). A happy accident! This further improves resolution down to 0.25µm. Almost twice the resolution achievable when using only white light illumination!
There are both advantages and disadvantages which I won't go into now (post is already too long) but in certain situations it's good enough to avoid switching to immersion objectives and the related inconvenience of using oil. I'm happy with that!
Amazing, techy stuff! Did you measure the pore distance within the areolae?
René
Hi Steve,
ZitatThe objective is not corrected for UV so I had to move it closer to the specimen to get focus. This lets the lens collect more, steeper-angle (higher NA) rays from the specimen, effectively increasing its NA to 0.85 (or more).
I don't think there is a significant increase in numerical aperture due to longitudinal chromatic aberration. We are talking here about a typical maximum deviation of the focal length of 1 % for fluoride objectives.
Hubert
Hi Rene and Lupus. Thanks for your comments.
Rene: I didn't measure the diatom above. My resolution figures were derived by calculation and backed up by a previous experiment with Amphipleura pellucida and the same lens (see below).
Lupus: I fully understand your scepticism but empirical results say otherwise. I had to move the objective closer to achieve focus in UV light. Working distance was reduced (by far more than 1%) allowing higher NA illumination rays to enter. I didn't consider focal length, but I'm not sure it's relevant anyway. Longitudinal chromatic aberration is no concern as I'm using monochromatic light (365nm).
There *is* a marked increase in spherical aberration visible in the deeper parts of the specimen above. That's one of the disadvantages I didn't discuss.
Below is a full-field image of a strew containing A. pellucida. It's a single shot taken with the same 40/0.75 objective. The lighting is different: a Heine condenser set for a darkfield effect. I couldn't quite reach full darkfield so there's a hint of annular illumination which shows as a slight brightening at the centre. I don't believe this affects resolution though, only contrast.
The other image is a hard crop into the darkfield one, inverted to negative to show the resolved punctae more clearly.
As you can see the dots *are* just resolved, which cannot be explained by using UV alone (with an NA 0.75 dry objective). Given the typical spacing of striae and pores on A. pellucida, I could only explain the resolution achieved if the NA of the objective were increased to at least NA 0.85. Other people more knowledgeable than me confirmed objectives used at smaller working distance than they're designed for *do* operate at higher NA (if the NA of the illumination allows it).
If you can describe another mechanism that makes this possible, I'd be delighted to learn of it.
Thanks
ZitatI had to move the objective closer to achieve focus in UV light. Working distance was reduced (by far more than 1%) allowing higher NA illumination rays to enter.
The working distance is only a small fraction of the focal length. The focal length is measured from the so-called principal plane, which is far inside the objective in the case of more complex designs. The very small working distance of high-aperture lenses then results in a seemingly large relative change. However, the reference value that determines the NA is the focal length.
ZitatLongitudinal chromatic aberration is no concern as I'm using monochromatic light (365nm).
Longitudinal chromatic aberration is the actual origin of focal length reduction at the UV wavelength, compared to e.g. green light.
The theoretical resolution that can be attributed to a particular NA is not so definite, especially for phase objects. Effects such as the refractive index of the medium, the type of illumination, the image contrast due to the camera sensor and image processing also play a role.
For example, a fluoride lens does not have a constant wavelength-dependent focal length reduction at shorter wavelengths, but typically a maximum for light beams with high NA, a minimum at medium NA (but not necessarily with every design). This means that when focusing on object details produced by light beams with high NA, the other light beams of lower NA tend to be out of focus. This may possibly - at least theoretically - have a contrast-enhancing effect like ring-shaped illumination. But I don't know the specific characteristics of the Olympus objective.
Hubert
OK, I understand all that and thanks for taking the time to explain.
But I still can't understand how an NA0.75 lens can perform so far beyond the "expected" resolution - except by using my initial conjecture (or mental model) about what's happening. If I had a UV-corrected 40/0.75, I could probably settle the issue by direct comparison - but I don't, so I'll just have to accept that it demonstrably does something that's useful to me and leave it at that.
Thanks again for your time, and patience.
Cheers
Beats