dinoflagellate in cool colors

[René]

Hi guys, here's last days fun part of the work day:

This is a marine flagellate, and I admit it is not really looking like a happy cell...


Focussing at the surface gives a hint of what it might be:


It is stained with a fluorescent brightener, one that makes your wash whiter then white (or so the slogan goes)  8)
This way the thecal plates of this so-called armored flagellate stands out, otherwise I probably wouldn't have spotted it in the sample:



Sample from the Wadden Sea, stack of 11 images with 20x/0.7. Width of the cell is 20 um.
Identification to species is tricky, but the shape of the pore on top is a give-away for Scrippsiella.

Here's one such pore, top view:


With this pore it can attach itself to a substrate, as the start of encystment.

Best wishes, René

[rjlittlefield]

Very interesting! I do not recalling seeing the term "fluorescent brightener" used for staining specimens like this. Do I understand correctly that this stain absorbs in the UV and possibly violet, and emits blue?

Also, I am curious about the magnification. 20 µm at 20X is only 0.4 mm. That would be a very small area on most sensors. What type camera are you using, and are there other lenses in the system?

--Rik

[René]

Hi Rik, the stain is acually called Calcofluor White, commonly used as an additive in the paper industry and in laundry detergents. It binds specifically to cellulose and does indeed also work with violet/blue excitation, for example with a fluorescein filter cube (ie with green emission). But I like the UV filter cubes, as it gives 'true color' and I can mix in transmitted light while scanning my samples.

The images are at 100%, slightly contrast enhanced. C-mount camera: TIS cmos 5Mp, coupled via Olympus PMTVC (0.3x) and 2.5x NFK eyepiece to an IMT-2 inverted scope. There's a magnification changer, set at 1.5x. So total relay factor is 1.125x.
Yes, it is pretty small, but the objective is pretty sharp (20x NA 0.7 splanapo), at least sharper than my eyes can handle. Most of my stuff is small, the TIS cameras work nicely at reduced size (middle 640x480p of the whole field), generally used at 4x averaging, which effectively filters out pretty much all noise while still maintaining smooth image flow.
BTW, image stack is made with Helicon, 'Pyramid' method. It works great for fluorescence stuff, but it gives slight vertical stripes in the image, no idea why.

Thanks for interest,
René

[Ecki]

Rene,

this is a jolly good idea to use calcofluor to show the structure of the armor. Very nice image! I will try this too when I find some dinoflagellate this spring.


Rik,

when we were younger and went out dancing, there were so called black lights. Those made white cotton shirts glow in a bright blueish white. Saturday night fever That is the same effect as the one Rene showed. Calcofluor binds to cellulose and chitin. I use it to color fungi.

Regards
Ecki

[curt0909]

Rene,
these are very nice. Super sharp for a 20x objective at this magnification. I bought some 'whitener' a while back but haven't tried it yer. If its not too much trouble could you tell us how you go about the staining process?

[René]

Thanks Ecki, but it's not my idea. It's in use for some time to precisely typify armored dino's. But I perfected the procedure pretty much so that it is practically in routine use here.

Today I was working on a large saline lake in the delta area of the Netherlands, it is a funny place with very typical species, really different from the rest of the delta.



Its looks like Gonyaulax polygramma, but as always in this place they look slightly different from everywhere else, considerable smaller (this one 20 um width) and the large spine is also not very typical. Fun place for us in any case.

Then I came across this splitting couple:



And this is different, it is proper division unlike that Scrippsiella before that just burst out of his jacket at the weakest point.



It doesn't really look nice, after iodine preservation and a bleaching with thiosulphate I admit.
But if you check the fluorescently stained theca, you can see how controlled the break is over the entire half of the cell. Even the girdle lists are still intact.
One end gets the horn, the other the spine. This kind of division is specific for a group of armored dinollagellates, it's called desmoschisis.




Greetings, René

ps, Curt, you got a pn.

[Charles Krebs]

René,

Great technique, and as usual, very interesting discussion.