Some phase contrast (and pseudo-darkfield) images of cleaned diatoms

[Hobbyst46]

A year ago, I collected diatoms from wet beach sand, cleaned them with mild chemicals (SDS, EDTA, some H2O2), which left many of the frustules intact, and stored them in water:ethanol. I recently mounted them in Pleurax. Striae of some of them are better visible in this medium than in Norland 61.

Images were taken with the 100X1.3 Planapo Oil Ph3 phase contrast objective. Either with or without green filter. And an inexpensive USB eyepiece camera. According to my previous tests with a stage micrometer, chromatic aberrations are negligible, apart from the periphery of the image (albeit so verified only with 10X-40X Neofluar dry objectives). The colors in images 2 and 3 are due to the complex interaction of light with the diatom, which appears to attract current intensive research. Green light is usually considered to be optimal for phase contrast, but IMO the colors are prettier...

Two of the images were taken under green filtered light. Diatom identifications are pure guesses. The center diatom on image 4 is, I think, centro-symmetrical, but is it an octagon or other polygon, or a circle that carries protrusions?

[75RR]

Very nice! Particularly like the first and third images.

The fourth looks interesting. Perhaps worth a revisit at some point?

[Hobbyst46]

Thanks 75RR!
99+% of all marine diatoms that I have met are pennates, only a handful are (perhaps!) radial, because they are actually fragments. I will try and revisit this one with different illuminations.

[einman]

very nice.

[Hobbyst46]

Thanks einman.

[MicroBob]

Hi Doron,

nice images! The round one may be something like plate 5, nr.2 here: http://www.deepseadrilling.org/13/volum ... 2_34_5.pdf

Bob

[Hobbyst46]

Thanks Bob for the identification and the document. This and a web source on diatoms from Crete, Greece, Dalmatie etc are my current information sources.
So it must be round and not polygonal. Time to find where have all the radial ones gone...

[MicroBob]

Hi Doron,
I once borrowed diatom material from Oamaru for a member of out group. I just needed it to have nice objects to show measuring methods and show the limits of the attainable precision. I wasn't impressed when I found mostly dust and bits and the very occasional intact diatom. When I looked closer I found bits of ca. 0,5mm diameter round diatoms, beautiful too, but not a single intact one. So may be they are just quite fragile and are the first to break.
I was in Croatia in fall of 2017 and the water was extremely clear there. This was beautiful but there was also nothing to be found with a plancton sieve. .

Bob.

[Hobbyst46]

Very nice! Particularly like the first and third images.

The fourth looks interesting. Perhaps worth a revisit at some point?

I probably misprinted the correct location of the item, and could not retrace and find it again. It is lost.

Luckily, while re-exploring my strew slide of marine diatoms in Pleurax, found some other pleasing items which I would like to share, if no one objects. I plan to post them later on.

But I will start with two unknowns. What are they ? diatoms ? fragment of diatom ? Skeletons of non-diatom organisms ?

The first, imaged with 40X0.75 Neofluar Ph3 (photo 1), a 50 micrometer size sort of heart shaped ornamented object. Not really radial, but more like a circle than and ellipse. The pattern is of relative coarse "gage".
My survey was done with the 100X1.3 Planapo oil Ph3. Possibly exerted some nano-pressure on the coverslip. Because, I returned to the "thing" afterwards, and it had moved away from the pile of diatoms where it had nested before (photo 2, 40X0.75). I believe that it is not alive, so Pleurax is not so rigid ? even after 15 min at around 180C as per the instructions ?

The second, more humble, is a "C" like form (photo 3, 40X0.75).
Any ideas would be welcome.

[MicroBob]

Hi Doron,
have a look on page 29 middle left: This looks like your first big diatom. http://www.mikrohamburg.de/Goeke/Diatomeen_gesamt.pdf.
They seem to have a potato chip shape.
The second one - no idea. When you haven't applied a strong acid it might be a calcareous structure completely different from a diatom.

Bob

[Hobbyst46]

Some more marine diatoms, from the same strew slide, mounted in Pleurax and imaged with the 100X1.3 Planapo Ph3 oil objective (and achromat-aplanat condenser, NA 1.4, oil) under phase contrast. Photos are either single images (photos (4) and (8)), where I thought that stacking did not contribute, or stacks of 5-11 images. USB eyepiece camera, controlled with Micam software. The contrast was enhanced in the control software menu. This enhancement created the dark background, which I nicknamed "pseudo dark field". The only post-processing was resize to fit upload limitations.

For comparison, a true darkfield single image was recorded with the 40X~0.8 Planapo oil (iris) objective and the darkfield position of the turret condenser (oiled to the slide as well). This image, photo(8), was cropped by a factor of 2.5 to equalize the diatom size to that of the phase contrast image.

As far as I can see, many frustules are fairly intact, and not many girdles spread around. I believe that this result is owing to the mildness of the cleaning procedure, however, these claims are not as yet substantiated.
Diatom identifications are tentative, will appreciate corrections if any.

[75RR]

Again very nice. Do like 2,3 and 4

[Hobbyst46]

Thanks a lot 75RR.

[Hobbyst46]

Exploring a strew slide of self-collected and cleaned diatoms revealed, among hundreds of very familiar pennate forms, some surprizes. Photo 9 - Petroneis pendant on a chain (what is this chain made of?), recorded with an eyepiece camera; photo 10 - a Gyrosigma, not a sharp image although the stria are visible; photo 11 - a colorful fragment of some centrales; photo 12 - a tiny, yet centrales cobwheel diatom, photo was cropped by about 2X. Photos 10-12 recorded with canon camera.

I believe that many of frustules are quite intact, probably due to the the mildness of the cleaning. Hence the valves are not optimally laid for photography, as previously mentioned by experienced microscopists previously on this forum.
All comments are welcome.