acridine orange stained live tumor cells

[Leo van Griensven]

Dear All,

The cells shown here are live K562 cells stained by ethidium bromide and acridine orange and immediately upon staining photographed in a Zeiss Axioskop usi Zeiss Axiocam and Zeiss software. Excitation 485nm , emission 538 nm. Objective Zeiss Neofluar Planachromat 40x.
K562 is a immature erythoid leukemia cell that has been in culture for several 100 passages.

Acridine orange is a vital stain that binds to nucleic acid as can clearly be seen from the prominent nucleoli like structures. Even a mitotic figure is visible. When taken up in the acidic environment of lysosomes it will have a bright orange color. Ethidium bromide will only penetrate in dying or dead cells and is specially used to study apoptosis. The cells shown here are vital and only one is red (and dead).

The cells are rather weak and after a while between objectglass and coverslip they flatten and make large extrusions. Pancake like I would say.

Something is definitely wrong here: I see these AO stained cells bright green in the microscope. The very beautiful Zeiss Axiocam and its software make the cells however blue. It looks nice, but it is wrong and I cannot solve the problem

Does this look familiar to some of you ?

Thanks,

Leo van Griensven

[Heribert Cypionka]

Code Auswählen Erweitern

It looks nice, but it is wrong and I cannot solve the problem

I know a little freeware tool that could help you... ;-)



Apart from that, we are using Axiovison (don't like it too much) , but never had this effect.


Best regards,

Heribert Cypionka

http://www.picolay.de

[Leo van Griensven]

Code Auswählen Erweitern

It looks nice, but it is wrong and I cannot solve the problem

I know a little freeware tool that could help you... ;-)



Apart from that, we are using Axiovison (don't like it too much) , but never had this effect.


Best regards,

Heribert Cypionka

http://www.picolay.de

Thank you very much, Heribert. This will help me.
I will look further though what causes the effect like some small error in the settings.

have a good day,

Leo van Griensven

[Ole]

Hello,
I faced a similar problem when analyzing tonsils (FFPE samples) stained with acridine orange. First the results looked like expected (green-orange). However, after performing an automatic correction of the white balance, the colors changed "irreversible" to blue-orange (camera: Leica EC3). Maybe this might provide a hind to solve your problem.
Regards,
Ole


Before...


...and after automatic correction of the white balance.