Hi all, as promised here a compilation of some images of Mallomonas teilingii.
A colleague of mine was fishing for cladocerans in a a pond near Lelystad (NL), with this winter-blooming chrysophyte as bycatch. After him filtering off his thingies, I got the rest of the sample for experimenting.
The chrysophytes are generally identified by the structure of the scales. Like with diatoms, the scales are largely silica based. And even though those scales are not *very* small (as in, the size of the smallest fraction of diatoms), they are very thin and thus hard to see. Normal mountants are out of the question. But water can be used, as the difference between the refraction index of the silica scales (ca 1.45) and that of the mountant (water, 1.33) is just about enough to be useful. In differential interference contrast that is. It does need oil immersion though, the image with the dry 40x/0.95 planapo is only just about enough to say there's some structure there, but not much more.
Images with Olympus DICT-S system, dry 0.9 NA condensor.
Best wishes, René
For scaling of this reticulate structure of the bodyscales, think slightly bigger than Pleurosigma angulatum!
So how to get the most out of these structures? They need quite a serious contrast boost. Currently, dry-mounting on a coverslip is the most effective method, but that does limit the resolution to that of a dry (<1 NA) objective. So, like with diatom mounting, you can make use of media with high refractive index to gain the most contrast from the specimen. Pleurax is my favorite, because it allows me to mount whole cells without them falling apart. The through-focus stack here is made with the standard Olympus DIC (DICTS) with dry 0.9 NA condensor toplens.
Now, I do also have to that ultimate oiled 1.4NA DICT-HR kit. Quite disapointing however: I couldn't make use the full condensor aperture, the image looks very washed-out. Nevertheless, the image benefits from condensor immersion, even at lower aperture. See for the comparison images on https://www.mikroskopie-forum.de/index.php?topic=40111.msg296009#msg296009 (https://www.mikroskopie-forum.de/index.php?topic=40111.msg296009#msg296009)
Best wishes, René
Thanks for the sharing and effort Rene, the "beast" was new to me.
Dank je Maarten, certainly from SEM images it looks like Smaug. Where's the place for a harpoon to get through this armour? ;)
Following images are made with a Phenom table-top SEM (lucky me). The silica scales show up as a quite simple baseplate with a reticulated pattern on top. The long 'bristles' are connected to the scales front and back by a spoon-like hook. There's no differentiation in the scales, they are all the same, even if they carry bristles.
Every cell should show a flagellum, but it's sometimes a bit difficult to find it among the bristles. The flagella of chrysophytes pull the cell forward. To help with that pulling movement, the flagellum is covered with fine flagellar hairs (insert).
Technics: glutaraldehyde fix (1%Glu+ 50mM HEPES), via ethanol and hexamethyldisilazane air-dried on small coverslips. Sample was sputtercoated with gold for conductivity. Gold is excellent for signal but unfortunately quite coarse. You can see the deposition structure in the background of the flagellum image.
Best wishes, René
Hi René,
Fantastic images and preparations, both for LM and SEM. I also very much appreciate your brief comments on differences in refractive index between scales and mountant and what this means for visibility in the light microscope. In a way your LM images are very encouraging - I conclude that I may keep on with my N.A. 0.9 dry condensor on my BH-2 (I sometimes have my doubts about this when Martin shows his magnificent images - which, by the way, demonstrate beyond any doubt that an immersion condensor N.A. 1.4 immersed at least with water is essential when you want to image minutest detail. Providing, however, you get the sample preparation right, careful prior isolation of specimen, thickness of mountant ... ).
Anyway, great microscopy, thanks a lot!
Best wishes
Ole
Hallo René,
sehr schöne und gelungene Bilder! Die REM-Aufnahmen gefallen mir besonders. So ein Tabletop-REM braucht ja kaum mehr Platz als ein größeres Lichtmikroskop, wenn nur der Preis nicht wäre.
Erstaunlich finde ich, dass die Schuppen offenbar so dünn sind, dass sogar der Elektronenstrahl durchgeht und die darunterliegenden Geißeln sichtbar werden.
LG
Jürgen
Zitat von: jcs in Februar 23, 2021, 21:46:34 NACHMITTAGS
Erstaunlich finde ich, dass die Schuppen offenbar so dünn sind, dass sogar der Elektronenstrahl durchgeht und die darunterliegenden Geißeln sichtbar werden.
Hello Jürgen, what you actually see is mostly the gold conductive layer, shaped by the specimen structure. I can play with it, by changing to higher kV (more penetrating power), or using another conductive coating. Chrome for example gives a very fine grain compared to goldsputtering. But is not as heavy, and therefore less efficient than gold for imaging. Next image shows a coating of 6 nm chrome with 10 nm carbon. The scales are almost translucent compared to the gold coated sample, and you can easily see the underlying scales. I's a neat trick!
Best wishes, René
Rene, thanks for sharing, I enjoyed your report very much!
Viele Grüße
Jochen
Hallo Rene,
Een intrigerend organisme en mooie beelden. Groeten,
Rolf
great! thats where we living for :-)
Wolfgang