On Mon, 18 Sep 1995, Matthew Hannah wrote:
 
>
> Doesnt this compromise increase the possibility that there will be bleed through
> from one channel into another? In my experience (which is with FITC/LRSC double
> labelling, I have no experience of Texas red) in most instances where you really
> need to show that an antigen is, or is not co-expressed in the same compartment
> it is necessary to excite the fluors separately .
 
 
The FITC signal does bleed into the red channel whether you do single
line or separate line excitation because the tail of the FITC emission
spectrum overlaps that of the Taxes red.  You may not notice it when you
excite them separately because both signals are stronger and the 'tail'
signal doesn't show up.
 
We are currently experimenting a FITC/cy-chrome combination. Both are
excited at 488 (no need for Ag/Kr laser).  Cy-chrome emits at 670, beyond
the tail of the FITC emission.  With a 645 dichoric and a 655 barrier
filter to stop the green from bleeding to the red, we found no bleeding
over at all even at maximum gain.  However, we are not 100% happy because
the red signal is weak.  Perhaps the 665 is too high as a barrier  (or
should we blame it on the quality of the dichoric ?) Could someone shed
some light on this?
 
---------------------------------------------------------------------
Harry Leung,
Technical Officer, Electron Microscopy & Confocal Microscopy,
Zoology Dept., University of Western Ontario, London, Ont., Canada. N6A 5B7
Tel: 519-679-2111 x6733  fax: 519-661-2014
E-mail: <[log in to unmask]>
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