Diatoms in Circular Oblique Light Update 06/05

[Charles Krebs]

Fantastic!. The clarity really surprises me for circular oblique. Do I understand correctly... you used a 0.8 NA dry darkfield condenser? (In addition to the lens iris and condenser height).

[pierre]

Very impressive work

Many thanks for opening a bright path in the diatom's illustration

[WalterD]

Dear all, thanks again for the kind remarks!

@James: The 100x objective has got an iris diaphragm which was partially closed. According the specs the Na with full open iris diaphragm is 1.32, closed 0.6. In this setup it is more open than closed.

@Charles: Correct, a dry 0,8 Na darkfield condenser is what I used, the 1,20 Na type just did not give the best results. The amount of detail is more than I expected with this 0,8 Na, I will crosscheck with a calibration slide what actual detail level can be measued in this set.

Five more pictures I've added:

Note: picture 16 was done with inverted desaturated DIC.


16 Rhaphoneis amphiceros
17 Cocconeis pediculus
18
19 Coscinodiscus radiatus
20 Hyalodiscus laevis

[Smokedaddy]

@James: The 100x objective has got an iris diaphragm which was partially closed. According the specs the Na with full open iris diaphragm is 1.32, closed 0.6. In this setup it is more open than closed.

... so your objective has a higher NA than the low NA of your dark field condenser? Exactly what objective is it if you don't mind?

-JW:

[WalterD]

... so your objective has a higher NA than the low NA of your dark field condenser? Exactly what objective is it if you don't mind?

James, indeed the objective has a considerable higher Na than the condenser.
The objective is, to be more precise, a100x magnification Leitz PL Fluotar oil immersion objective with an Na of 1.32 with full open diaphragm and 0,6 with the smallest aperture. ( an image is also shown on the previous page) The objective is designed for a mechanical 160mm tube length and 0.17 mm coverslip thickness. Although my Leitz Orthoplan microscope has a tube length of 170 mm, this does not seem to downgrade the image quality in this setup. The iris diaphragm provides a lot of opportunities for darkfield and COL at this magnification.

Regards,

Walter

[Robert Berdan]

Interesting images with a 3D look and looks like you are achieving very high resolution - they almost look like bone fossils.

Nice job
RB

[WalterD]

Good to hear that, Robert.

Now what resolution did I achieve? The angular resolution formula is helpfull to assess what could be expected . (https://en.wikipedia.org/wiki/Angular_resolution)


The partially open iris objective has an Na of something like 1.1 (not 1.32 when fully open) and the dry darkfield condenser 0.8. The average wavelength of white light is 0.5, so the formula is completed as folllows:
R = (1.22*0.5) / (1.1 + 0.8 ) = 0,32 micrometer. So any detail of 0,32 mu would just be visible, according this formula.
To check this I used the Hyalodiscus laevis picture. My camera frame is 220 mu wide at this magnification and the diatom itself was found to have a diameter of 105 mu.



The diatom itself is described as having between 28 to 45 pores per 10 micrometer, each pore 0,1 mu wide. This can also be seen on SEM images.
https://www.fottea.czechphycology.cz/pd ... /02/13.pdf Below crop is 10x10 mu.



In the detail I cropped, a 2 micrometer square, rows with 5 pores can be seen. So that is slightly less compared with the a.m. 28 pores. (23 pores per 10 mu actually)



In case the pores are also 0,1 mu wide in my slide, this resolution was achieved with this COL (circular oblique light) setup. In case the pores are a bit wider in my slide the resolution is at least more than double than according above resolution formula. Am I right or did I overlook anything?

[Soki]

Superb quality. The 3d effect is amazing.

kind regards,
Simon

[René]

Hi Walter, great images again! Number 18 is Trachyneis aspera, a beautiful and common diatom in the North Sea.

The definition for resolution is the abilty to resolve two points at the said distance. And it is only an estimate as it depends on the structures and the type of illumination.

Best wishes, René

[rjlittlefield]

Walter, the images are excellent!

... the resolution is at least more than double than according above resolution formula. Am I right or did I overlook anything?

Explaining more about René's point, the R number would be, for you, the distance between pores in a pore/space/pore/space sequence. In your 2 micron square, I measure pore-to-pore distances in the range of 0.42-0.45 microns, versus 0.32 that the formula predicts you could just barely see.

So, your imaging is doing great, but no, not better than the formula predicts.

--Rik

[WalterD]

Soki, Rene and Rik: Thanks for your kind remarks!
@Rene and Rik : I get the point regarding the resolution query. So the formula does not need to be rewritten

[jmc]

I know this is a couple of years old now, but what amazing images. Thanks for sharing.

[WalterD]

Hi Jonathan, thanks for your positive feedback.
Time flies, I remember doing this when I had more spare time at the beginning of the pandemic.
It may be an idea to also take pictures of the remaining diatoms of the slide though, as I did not complete all 104.

Nice diatoms job you're doing as well with your most recent post!

[grgh]

Sadly for all who knew Klaus Kemp, either through personal contact or as an admirer of his work.
Received the news that he passed away on the 28th May 2022 in Hospital.
In his passing, the world of micro-manipulation has lost a great master of the art.
R.I.P

[jmc]

Thanks Walter. Funnily enough, I only got into microscopy in March 2020 as a result of the pandemic - work dried up. I bought a beaten up old Olympus BHB with the idea of fixing it up and learning a bit about a new imaging area. That ended up being my start point for the UV microscope build.

Grgh - yes very sad news indeed.