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Hello Ray

Do you have to use PE? Its spectral characteristics are really not very
good for multiple-labelling... especially with fluorescein. If you have
to use the PE, you could try a higher pass red emission filter, and you
may be able to get a signal just from the PE. I don't think there is any
way to get the PE signal out of the fluorescein other than by
subtracting a pure PE image, if you can get one. Presumably one of the
new spectral imaging systems would be able to handle this problem, but
I'd reckon it would be cheaper to get a new (non-PE) probe!!

IAN


ray hester wrote:
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi,
>
> If you have a monochrome cooled CCD camera and cells labeled with FITC and
> phycoerythrin, is there a way of imaging these cells and 'seeing' both the
> green (FITC) and orange (PE) labels?
>
> With a color camera, of course, even using only the FITC excitation cube,
> you see both green and orange.  But with the monochrome camera, all of the
> fluorescence (FITC _and_  PE) looks similar (before assigning colors) and
> there's no way of knowing to what structures to assign green or orange.
>
> Since both FITC and PE excite at 488, but PE excites better at 514 (or
> whatever wavelength the TRITC cube selects) is there some way of subtracting
> the 514 excited FITC from the 514 excited PE?
>
> There must be some way of doing this.  Incidentally, we have MetaVue
> software.
>
> Thanks for any help.
>
> Ray Hester
> University of South Alabama
> [log in to unmask]

--
Professor Ian Gibbins
Anatomy & Histology
Flinders University of South Australia
GPO Box 2100, Adelaide, SA 5001
Australia

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