Ian Gibbons writes:

> There is a real problem emerging here that people on the list may like to
> comment on... As fluorophores, optical systems and detectors all become
> more efficient, getting complete discrimination between different
> fluorophores in multiple-labeled preparations is becoming more and more
> difficult with the current lot of labels. We recently got some very narrow
> band barrier filters made for our BioRad 1024 system, especially for the
> PMT detecting red fluorophores, so that there would be minimal fluorescein
> bleed through. Although in the end we would have been picking up just the
> very end of the emission spectrum of fluorescein, it still could be
> detected quite easily by the detectors set with relatively moderate gains.
> We also have seen this occur in our wide-field fluorescence microscopes.

Ian--

Is fluorescein showing up through your rhodamine PMT when exciting only with 568
nm?  --That's what it sounded like from your message.  If so, something's
weird--this shouldn't happen under any real-life circumstances of which I'm
aware.  Any chance that your fluoreceinated secondary has been contaminated with
a bit of rhodamine, or that the excitation filter has a pin-hole light leak (or
some other flaw) in it?

Martin