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Hi,

I've run into some problems while trying to image samples containing Cy3
and Cy5-labeled proteins by TIRF.  If I image the Cy5-labeled components
briefly, and then look at the Cy3 with 514nm excitation, the Cy5-labeled
component is visible.  This happens even in samples that only contain Cy5.
It isn't a simple matter of cross-excitation of Cy5, bleed-through, or
FRET since the Cy5 only becomes visible under the Cy3 settings
after it's been exposed to 630nm light (for 1-2 seconds) or 514nm light
(for 20 seconds or more).  It seems like some photochemical process might
be converting the Cy5, or the protein it's attached to, into a chemical
species that fluoresces at 514nm.  If anyone has any idea of what could be
causing this, I'd love to hear about it.


Thanks,

Adam