High NA Diatom Images

[josmann]

Hey folks! I spent some time last night imaging diatoms off of a strew slide. Basically using a 1.4 NA objective with polarized, quasi-monochromatic COL. Got some images that I think are pretty darn good! I've shared a few low quality versions of my most interesting finds here, but please view the full quality in my GDrive folder so you can zoom in on poroids and lineolae and whatnot. More info on my setup in there too: https://drive.google.com/drive/u/1/fold ... mMKyW4_Dgk

[josmann]

I also wanted to share this video I captured. For this, I was basically in brightfield configuration but I had a polarizer over the field lens (no analyzer). As I rotate the polarizer (slowly at first, then faster later), you can see the lineolae of the N. oblonga I'm imaging appear and disappear based on polarizer orientation. This is a really interesting effect and I'm not sure if it's described anywhere! Anyone have an idea?

[MichaelG.]

Very impressive, and very interesting !

I suspect [and it’s nothing more than that] that the lineolae are effectively a diffraction grating and you are seeing them appear/disappear in the same way that a pair of polarisers interact.
Close study of your polarising filter might be worthwhile.

MichaelG.

[Hobbyst46]

Impressive !
I tried to see details of your microscopy setup but did not find them in the link.

[MichaelG.]

Impressive !
I tried to see details of your microscopy setup but did not find them in the link.

.

Have a look at the text file at the bottom of the list.

MichaelG.

[josmann]

Impressive !
I tried to see details of your microscopy setup but did not find them in the link.

EQUIPMENT AND ILLUMINATION: Olympus BH2 BHT modified with 100W LED. Light polarized at field lens and then filtered through green interference filter (GIF). Amscope oil immersion darkfield condenser (NA 1.36-1.25) for circular oblique light (COL). Cargille Type A immersion oil. Olympus SPlanApo 100x 1.4NA objective lens with aperture opened all the way. Polaroid film for analyzer crossed against initial polarizer for maximum extinction. 1.25X intermediate tube (DIC tube with DIC slider removed). Olympus trinocular head with 2.5x photo eyepiece. Slider set for 100% of light to camera. Sony FX3 camera with ISO set to 100, 2.5s exposure triggered with IR remote.

Very impressive, and very interesting !

I suspect [and it’s nothing more than that] that the lineolae are effectively a diffraction grating and you are seeing them appear/disappear in the same way that a pair of polarisers interact.
Close study of your polarising filter might be worthwhile.

MichaelG.

I agree that the polarizer orientation almost certainly has something to do with the lineolae orientation. I was thinking something along the line of maybe light polarized along the same axis as lineolae comes out still nicely polarized like that while light polarized orthognal to the lineolae gets "lensed" by them and that scrambles the polarization, leading to less coherence at the image sensor. I wish I knew my way around some wave optics simulation software....

If you guys thought that was cool, check this out. For context, I'm in a debate with a diatomist on the species of diatom in question and the nature of the pores in its striae. But in the midst of that, I adjusted my settings a bit and am now getting some pretty exceptional results. I switched out my 100x SPA to a 60x SPA and I removed the green interference filter. This creates sort of an ugly yellow/brown background, so I popped a wave plate on top of the field polarizer and tilted it just a bit to shift the hue. I present to you what I believe may be some of the finest microscopy ever recorded in 4K/60fps:

[Sure Squintsalot]

Very impressive, and very interesting !

I suspect [and it’s nothing more than that] that the lineolae are effectively a diffraction grating and you are seeing them appear/disappear in the same way that a pair of polarisers interact.
Close study of your polarising filter might be worthwhile.

MichaelG.

I agree that the polarizer orientation almost certainly has something to do with the lineolae orientation. I was thinking something along the line of maybe light polarized along the same axis as lineolae comes out still nicely polarized like that while light polarized orthognal to the lineolae gets "lensed" by them and that scrambles the polarization, leading to less coherence at the image sensor. I wish I knew my way around some wave optics simulation software....

If that were true, the lineola on one side would appear (almost) concurrent with the disappearance of the lineola on the other side. I parenthesized "almost" because the lineola on one side are not orthoganal to those on the other side of the long axis. I suspect that your rotating polarizer is removing glare by merely accentuating minor background polarization; note how the background also dims as you rotate the polarizer.

In the context of microstructure effects on transmitted light, you guys will appreciate this paper:

Structure-based optical filtering by the silica microshell of the centric marine diatom Coscinodiscus wailesii
https://opg.optica.org/oe/fulltext.cfm? ... &id=294214

The C. wailesii frustule valve, which possessed a quasi-periodic hexagonal pore array, exhibited position-dependent optical diffraction. Changes in such diffraction behavior across the frustule were consistent with observed variations in the quasi-periodic pore pattern.

We have demonstrated that the silica-based valve of a representative centric diatom species (Coscinodiscus wailesii) exhibits distinct tunable filtering effects produced by position-dependent diffraction. Furthermore, we have found that the wavelengths at which such effects occur are correlated to the diatom valve pore structure; that is, the wavelength of the band-gap is directly related to the periodicity of the pore pattern on the valve.

yeah...that camera is pretty awesome. Damn you!

[Hobbyst46]

Thanks, Michael. Thanks, josmann.
I have a DF oil condenser and a 100X1.3 objective and a green IF and polarizer and analyzer. Problem is, focusing
becomes difficult because the combination of all that in series darkens the FOV so much. Worth a second try though.

[MichaelG.]

In the context of microstructure effects on transmitted light, you guys will appreciate this paper:

Structure-based optical filtering by the silica microshell of the centric marine diatom Coscinodiscus wailesii
https://opg.optica.org/oe/fulltext.cfm? ... &id=294214

.

Many thanks for the link

MichaelG.

[josmann]

If that were true, the lineola on one side would appear (almost) concurrent with the disappearance of the lineola on the other side. I parenthesized "almost" because the lineola on one side are not orthoganal to those on the other side of the long axis. I suspect that your rotating polarizer is removing glare by merely accentuating minor background polarization; note how the background also dims as you rotate the polarizer.

If you look carefully, there does appear to be an "out-of-phase" resolution effect on the left side lineolae that's just more subtle. One thing I've learned doing this stuff is that it's common for only one small portion of the frustule to really behave nicely. Very subtle tilts and whatnot lead to dramatically different effects. You can see that, when I resolve the pinnularia poroids in the vid, many of the poroids in other striae blend together into continuous lines. Takes a lot of fine tuning to get these waves to beat on each other right.

Anyway, thank you for the feedback and analysis - please provide more thoughts if you have any - these help drive my experimentation. One thing that would be super nice to have (which I don't) is a rotating stage. I suspect we'd learn a lot! I can try to do it janky style, though...

In the context of microstructure effects on transmitted light, you guys will appreciate this paper:

Structure-based optical filtering by the silica microshell of the centric marine diatom Coscinodiscus wailesii
https://opg.optica.org/oe/fulltext.cfm? ... &id=294214

They had me at "supercontinuum laser" - I was looking for one on eBay the other day. No real luck :/ I really want to get into some spectral investigations of different organisms. Appreciate the link!

[josmann]

Thanks, Michael. Thanks, josmann.
I have a DF oil condenser and a 100X1.3 objective and a green IF and polarizer and analyzer. Problem is, focusing
becomes difficult because the combination of all that in series darkens the FOV so much. Worth a second try though.

I'd pull the GIF out - I honestly don't think it contributes much and light throughput sounds like it's limiting you a lot more than anything else.

I'm also using my B&L 15x ultra-wide eyepieces when observing directly - these help stretch the tiny features we're attempting to resolve although it also stretches out the brightness since they still have a FN of 20mm.

[Chas]

I wonder if some of the effect comes from light reflected downwards (say from the coverslip) and becuase of the polarisation this becomes a bit directional (?)
Is this something a bit similar?; (right hand page half way down):
https://archive.org/details/microscope0 ... 0/mode/2up

[josmann]

I wonder if some of the effect comes from light reflected downwards (say from the coverslip) and becuase of the polarisation this becomes a bit directional (?)
Is this something a bit similar?; (right hand page half way down):
https://archive.org/details/microscope0 ... 0/mode/2up

Chas, thanks so much for pointing me to this book! I just picked up a hard copy! I love old resources like this - it's amazing what techniques have been long forgotten. They're basically describing the technique I used, but I always wondered where it came from.

I will be doing some more polarization investigations in the future - one clue is that, at lower mags, N. oblonga shows blue structural color from its lineolae and this structural color can be modulated by turning the initial polarizer!

[Chas]

I love old resources like this

In the UK, on ebay, there are USB drives e.g. "220 Rare Vintage Microscope Books on USB Histology Microscopy Slides Science F5" I dont have this particular USB but a few years ago I bought a similar one and it still keeps me entertained!
Curiously there some other collection titles that are available only on CD (from the same seller).
However it takes a while to work out which of the old books are rewarding reading and which are not