We have tried the different versions of the Oregon Green dyes for
fluorescence immunohistochemistry. There is no doubt that they are bright
and relatively photostable. However, we also found that they seem to have
very broad emission spectra in practice, so that they showed significant
bleed through a variety of barrier filters that selectively allow
viewing red fluorophores such as Cy3, Texas Red etc, but which discriminate
very well against fluorescein deriviatives. There is always going to be
some overlap in these fluorophores - it may be that the Oregon Greens are
so strongly fluorescent that bleed through is simply more noticeable.

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.

What can we do about it? The easiest option is to use fluorophores well
separated spectrally eg fluorescein and Cy5, and hope that chromatic
abberations don't get too bad... We also have been trying out Cy2 as an
alternative to fluoroscein, since its emission spectrum is a bit shorter
than fluorescein, and, in theory, present less bleed through in red
barrier filters - I don't have the results of those comparisons yet.

It may also be worth pointing out that the 488 nm blue line is actually
quite good at exciting Cy3, especially in well labelled preparations, so
you can get cross-excitation as well! (Texas Red is not excited
significantly by this line).

So in the end, we recommend that everyone using multiple-labelled
preparations must make a series of single-labelled controls and explicitly
test for bleed through and cross excitation on a run-by-run basis...

Sorry for getting off the track a bit, but this might be helpful...

IAN

On Tue, 10 Dec 1996 12:02:56 PST, Janine Harrison wrote:

>     Hello Imagers:
>
>     Has anyone had hands-on experience yet with these new Oregon Green
>     Dyes from Molecular Probes, and would like to make a comment?  I.E.
>     are they really brighter and more photostable?
>
>     Thanks for any helpful input--
>
>     Regards,
>
>     Janine
>
>     **********************************************************************
>
>     Janine Harrison
>     Manager, Image Analysis Facility
>     ICOS Corporation
>     22021 20th Avenue S. E.
>     Bothell WA 98021
>
>     1-206-485-1900 ext 2318
>     1-206-485-1961 FAX
>     [log in to unmask]
>
>     **********************************************************************
Professor Ian Gibbins                         Flinders Microscopy &
Department of Anatomy and Histology            Image Analysis Facility
Flinders University of South Australia
GPO Box 2100 Adelaide 5001                    Centre for Neuroscience
AUSTRALIA
Phone:  +61-8-2045271
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