Diatoms: Pinnularia sp. "dotted"

[Beatsy]

Hello all,

At 185nm spacing, I present an image of the closest punctae (dots) I've resolved to date. It's a single shot (no focus stacking) taken with a 100/1.4 PlanAPO objective under 365nm UV brightfield illumination. The contrast was strongly enhanced in post by tone-mapping in Affinity Photo 2.0

I think it's a Pinnularia but I won't guess the species as I'll most likely be wrong. For context, it was in a small mount I made to test something else (also pictured below). The forms were attached to a 0.17mm coverslip with Debes' fixative and mounted in Darax. They represent some of the larger forms found in cleaned freshwater diatomite from Ongarato Valley, NZ. The "dotted" diatom is second from the top in the mount.

I still have a little way to go as my system theoretically resolves to 160nm, but first I must find a diatom with features spaced that close. One day... 

Cheers
Beats

[purkinje]

Wow!
Chapeau 🎩
(Which condenser did you use?)
Kind regards Stefan

[Siegfried]

Excellent work. I can get excited about it.
  Greetings from Siegfried

[Beatsy]

Thanks Stefan. I used an Olympus BH2-AAC Aplanat Achromat 1.4 NA condenser, oiled to the slide. Nothing fancy.

And thanks Siegfried. It's comforting to know I'm not the only one who can be excited by a few small dots!

Cheers
Beats

[deBult]

Well done:resolution performance at the extreme of what is possible in light microscopy.

Thanks for sharing,
Best Maarten.

[RainerTeubner]

Hello Beats,
 
 I'm another one who is fascinated by small dots, shown in excellent photos!

Kind regards!

Rainer

[Beatsy]

Hello again,

I've added this to the thread because it involves the same mount, same equipment, same capture methods and the same topic (extreme diatom dotting).

This time it's about the diatom at the bottom of the mount, Hantzschia amphioxys var stricta.

When I looked it up I found the statement, "Striae are indistinctly punctate". It means the lines are obvious but the dots are hard to see. So of course, I saw that as a challenge and confidently set about resolving the dots. But it didn't turn out as I expected!

The form is a little bit bent with the centre stuck to the glass, but the left hand side slopes down into the mount. Not much, only 250nm deeper at the far left end. Part A is roughly 150nm-200nm away from the glass, part C is practically touching it. I'm not sure exactly where the focus plane is positioned though.

A SEM image would probably solve this conundrum, but I can't find one, and I don't own a SEM.

As you can see, only tiny differences in the plane of focus relative to the dots makes them look completely different. I have no idea which is the "correct" appearance but think it might be B. Or maybe A. Surely not C, is it? I have been suitably humbled! 

What do you think?

Cheers
Beats

[Rene]

Nice one Steve. Carefull, it's easy to get afflicted with the victorean disease of dotteritus 

I focussed through Frustulia saxonica once, and counted at least 6 different 'sharp' planes with interference patterns, and all of them are right ;-)

B seems the sharpest one. Nevertheless, images is focussed on the fibulae on 'higher ground' in the mantle, so I don't think it is the right (middle) focal point for  imaging the pores. So I would say image A gives the truest image with a single row of pores between the striae.

BTW, the fibulae seem very regularly spaced, is that for the whole of the valve? Where's your material from?

Regards, René

[Beatsy]

Thanks Rene,

I think you're right. I've attached a crop from the first image I took and then forgot about. I was distracted by the visible dots in the Pinnularia which I hadn't expected to see (it was also in the FoV) and concentrated on that instead. Open it in a new tab to see a larger version.

The UV lighting alignment was a bit different in this first pic and clearly shows the dots between striae. Each of the striae have their own fibula in the central 2/3rds of the valve but then appear to skip one or two near the ends. I'll make a new mount with 2-3 more Hantszchia in it and do some closer inspection. I'll report back in a day or two.

The material is selected from some freshwater diatomite I cleaned - from Ongarato Valley, NZ.

Haha. GET afflicted with dotteritus? I was born with it! 

Cheers
Beats