🚀 go-pugleaf

On Sun, 28 May 1995, Natalie Bilyk wrote:
>
> ...............................  We work with FITC and Texas Red and
> usually get very good results.  One occaisional problem though - when
> scanning on dual green/red mode I sometimes come across a background
> staining that is excited by blue light but emits in the texas red
> wavelength.  This forces me to abandon dual scanning and scan only for
> green or red emmission separately, which isn't always desirable.  I'm
> curious to know if others have seen the same problem ?
>
> Natalia Bilyk
> Department of Anatomy and Human Biology
> University of Western Australia


Yes, very much so!

Bleed-throuugh from green channel to yellow (TRITC) red (Tx Red) or even
far red (Cy5) is common to all instruments I used that is both Zeiss and
BioRad. 488 excitation, when used in multiple-fluorochrome, simultaneous
excitation/emission mode causes the right-hand tail of FITC emission to
be detected by all red channels.
Cure?
Difficult so far.  Filter modification might help a bit BUT an
improvement, if any, is unlikely to justify costs.
Different fluorochrome should help such as Bodipy FL (Mol.Probes) that
excites at ca. 500 nm and emits at 512 nm might be a candidate. Before
buying a year's supply of the fluorochrome I would ask Mol Probes for a
comparison chart or even better, LEGIBLE graphs showing shapes of emission
curves of both products (i.e., FITC and Bodipy FL) to see how far they
trail into red AND if the difference justifies switching to a new
flourochrome.
If anyone gets more illuminated on this topic, I would appreciate to know!
Thanks
michal

Dr. Michal Opas
Department of Anatomy & Cell Biology
University of Toronto
Medical Sciences Building
Toronto, Ontario, M5S 1A8 Canada

tel 416 978-8947
fax 416 978-3954
e-mail m.opas@utoronto.ca