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I don't know the answer to this.  BUT it is clear that there is
some photobleaching / photoconversion going on and since your blue
signal doesn't decrease the implication is clearly that your
excitation is saturating the fluorochrome.  In other words,
you should be able to reduce the Hoechst excitation (405nm?)
very substantially without any loss of signal.  So cut down
your excitation until your emission starts to fall and you
should see a substantial improvement.

                                                Guy


Quoting Xinyu Zhao <[log in to unmask]>:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear listers,
>
> We were looking at a cell double stained with Hoeschst 33258 and Cy3.  At
> the
> beginning, the nucleus was beautifully blue in the blue channel and the red
> channel only had the red signal expected from the Cy3 lableling.  However,
> after serveral minutes, the nucleus started showing up in the green
> channel,
> and then in the red channel as well, beautifully red, too.  And at the same
> time, no observable change of signal level in the blue channel.  Similar
> things also happened with laser scanning in the past.
>
> Does anybody know why it was happening?  Is there a way to prevent it?
>
> Thank you very much.
>
> Jasmine X. Zhao
> Biomedical Imaging Core Lab
> University of Pennsylvania
> B110 Richards Building
> 3700 Hamilton Walk
> Philadelphia, PA 19104
>


--
Associate Professor Guy Cox
Electron Microscope Unit, F09
University of Sydney NSW 2006
+61 2 9351 3176