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Andrea Murmann wrote:

> i am using a Zeiss LSM 510, inverted microscope, single
> photomultiplier system with three color lasers: HeNe (633 nm) and
> Ar/Kr (488/568nm). I am interested in three dimensional multi
> color-FISH (fluorescence in situ hybridisation) on human nuclei. so
> far I am using 3 colors in following combinations: fitc/alexa488,
> rhodamine/cy3 and cy5.
>   i would like to increase the number of fluorophores detected at one
> given time.
> Do you have any suggestion, how i could increase my number of
> detectable fluorophores in my system? i was thinking to combine the
> three fluorophores with tandem-fluorophores as e.g. offered by
> Molecular Probes (see http://www.probes.com/lit/bioprobes38/6.pdf),
> but they are generally used for FACS-analysis.
> Does any one of you already have experience with tandem-fluorophores
> in FISH-applications?
> Could i use tandem-fluorophores and combine these with the other
> three fluorophores, and detect them on the confocal microscope in
> multitrack-function?
> Are there possible background problems/bleed-through?
> Could energy transfer occur from the single fluorophores to the
> tandem-fluorophores (in case of colocalisation)?

I personally have no experience with tandem fluorophores, but expect
that bleed-through WILL be a problem.  If you have a red photon, you
have no way of knowing whether it originated with your red fluorophore
or from the "tail" of the emission of a green fluorophore.  The easiest
way to be certain is to excite with wavelengths of light that won't
excite your green fluorophore efficiently--this is called "sequential
imaging" on our Bio-Rad 'scope.

That being said, I've seen citations to people out there who are pushing
the envelope by recording the emitted spectra and then deconvolving the
spectra (...as opposed to the image) to infer how many fluorophores are
present.  If you do a medline search for "spectral imaging", you'll
probably come across some of these papers.  I'm not sure whether any of
these folks are using confocal, but they might give you some ideas.

Good luck!

Martin Wessendorf
--
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