freshwater dinoflagellate

[Rene]

Hi guys, here's one of my projects, an undescribed freshwater dinoflagellate of the genus Durinskia.

(all images 50x50um, with 20x/0.7 objective)


I've fixed cells with ethanol, and stained them with DAPI, a powerful fluorescent DNA stain. It stains the large dinoflagellate nucleus brightly, and it also shows that it is organized in little threads (chromosomes). That is common in dino's, which have such an absurdly high amount of DNA that it is permanently in a condensed state.



Posting these images has been triggered by the discussion on DNA content of the chloroplasts and why it is so hard to show its presence (on Paramecium, https://www.mikroskopie-forum.de/index.php?topic=25818.0). I've always thought DAPI to be very sensitive. Might the DNA content of endosymbionts such as chloroplasts be too little for detection with DAPI?

Best wishes, René

[JB]

Hi René,

The DNA content of plastids is quite low compared that of the nucleus. In addition, the photochromes absorb and quench excition and emission light. To separate the strong fluorescence signal from the nucleus and the weak signal from the plastids, you best physically separate the plastids from the rest of the cell, either by extracting the plastids or by at least by making a squash preparation. Also remove excess DAPI from the mount (no DAPI should be left in the mountant for best results).

My suggestion:

Fix.
Wash.
Stain.
Wash.
Mount and squash.

If that isn't enough, consider extracting the photochromes with an alcoholic fixation solution before staining.

Kind regards,

Jon

[Oecoprotonucli]

Hello René,

it's always nice (and useful) to see the Nucleus/DNA in relation to the rest of the cell!

Best regards

Sebastian

[Rene]

Thanks for the advice Jon.
Unfortunately the blooming time is over, and I haven't got fresh material anymore. So I've used some cells that I have fixed with ethanol/NaAc on 19-4-2016 (which was used for the first set of images!)
Not surprisingly, it seems that the autofluorescence is slightly higher in the cytoplasm. The DAPI stain remains very strong in the dinokaryon, but I cannot detect any staining that I can attribute to chloroplast DNA.


brightfield and fluorescence, (UV excitation and long-pass filters), RGB channel 20x/0.7


Fluorescence: blue channel only; blue and red channel. Funny to see DAPI still gives some red fluorescence that shows up here. Of course the blue channel is overexposed severely.

OK, no succes this time, waiting for the next possibility with live material.

Best wishes,
René

[Rene]

Thanks Thilo. The article by Coleman gave me hope to be able to do see chloroplast DNA. Until now I only saw intricate confocal images.
I suppose freshly fixed material is key to start with: fixed material quicky starts to develop aspecific autofluorescence.
Do you know whether Hoechst dyes are better in terms of fading or detection level? Wikipedia only mentions differences in cell permeability.

Best wishes, René