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I think I understand the principles behind spectral unmixing, but I'd
appreciate some clarification. Just so I completely understand the
technology, (my brain likes simple so as not to be seduced by the "dark
side" (good one!!)), would it be fair to say that "spectral unmixing" or
"lambda scanning" is just "fancy cross-talk correction"? I suggested this to
a rep and he was most indignant and said it wasn't, but didn't explain very
well how it differed. Am I wrong and misunderstanding this or was the rep
just upset at my disdainful tone!?
Thanks
Tony
PS. Can it *really* discriminate detween GFP and YFP in a "real" experiment?

Research Associate
Laboratory of Molecular Signalling
Babraham Institute
Babraham
Cambridge, UK
CB2 4AT
Tel: +44 (0)1223 496499
Fax: +44 (0)1223 496043

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> -----Original Message-----
> From: Stephen Cody [mailto:[log in to unmask]]
> Sent: 01 May 2002 02:10
> To: [log in to unmask]
> Subject: Re: Carl Zeiss LSM 510 Meta
>
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear List,
>
> Owen Schwartz wrote:
> >You can use reference spectra that are supplied by Leica, or
> collect your
> own >lambda scans ....
>
> I should mention that I don't have either a Leica SP2 or a Zeiss Meta
> system. However, at a microscopy meeting I had some good
> debates with the
> Zeiss people about spectral unmixing. It occurred to me then,
> that a danger
> exists that people may use predetermined libraries of spectra to unmix
> spectral overlap with their own samples. I believe that this
> will often give
> erroneous results, as the spectra of dyes change according to the
> environment that they are in. Spectra measured in solution
> may be quite
> different from spectra measured in one cell or another. I was
> told it was
> for this very reason that Zeiss did not supply reference
> spectra, as they
> felt that every researcher should generate their own. I would
> suggest that
> individual machines may also vary so that the spectra may
> appear different
> when measured on a differently calibrated machine, which may
> account for
> Robert Zucker's question.
>
> I would take it even further. I wouldn't be surprised at all
> if you measured
> the spectra of a dye within 10 different cells from the same
> culture you may
> get 10 slightly different results. I made the suggestion to
> the Zeiss Rep.
> that a spectral averaging facility would be useful, so that
> the researcher
> could at least generate their own average spectra as a reference.
>
> Those that are experienced in calcium imaging know that it is not good
> practice to use a calibration reported in a previous paper. Dye
> calibrations, on the same instrument, with the same cell type
> (if not the
> same cells) should always be done.
>
> Unmixing spectral overlap I'm sure will be a very useful tool, but we
> shouldn't be blinded by the technology and forget good science. As my
> supervisor once said regarding a similar issue ' "Beware the
> dark side",
> don't be seduced by the technology.'   :)
>
> Stephen H. Cody,
> Colon Molecular and Cell Biology Laboratory,
> Ludwig Institute for Cancer Research,
> Post Office Royal Melbourne Hospital,
> Parkville, Victoria 3050, Australia.
>
> Tel: +61 3 9341 3155 (BH)   +61 3 9341 3158 (@work AH)
> Fax: +61 3 9341 3104
> email: [log in to unmask]
> http://www.ludwig.edu.au/labs/confocal.html
>