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 FAX: +61-8-2770085 e-mail: [log in to unmask]