On Thu, 15 May 1997 10:32:49 +1100 immunogenetics
<[log in to unmask]> wrote:
  The laser is a Argon Ion Laser with two laser lines 488 nm and
> 514 nm (It is also capable of running 50/50).  Two channels can be run
> simultaneously with a splitter which allows 510-550 nm to channel 1 and above
> 550 nm to channel 2.  Channel 1 can be filtered with two long-pass filters at
> 515 or 530 nm and Channel 2 can be filtered with two long-pass filters at 550
> or 590 nm.
>
> I would like to do two colour fluorescence looking at the co-localisation of
> two proteins.  I was wondering if anyone has a similar confocal setup and what
> fluorescent dye combinations are appropriate for the above filter sets (with
> minimal bleed through).  The fluorescent dyes need to be conjugated to
> antibodies.
>
> I have already tried FITC and Rhodamine B ITC.  With this combination I have
> great FITC signal with the 515 nm Filter on channel 1 but there is bleed
> through of the FITC into Channel 2 with the 550 nm Long-pass filter.  If I put
> the 590 nm Long-pass filter into Channel 2 I eliminate the FITC bleed through
> but eliminate most of my Rhodamine B ITC signal.

FITC has a very long tail in its emission spectrum when
excited at 488, extending above 590. (See Fig. 1.10 in the
Molecular Probes catalog).  Thus, if you have very bright
FITC, it is impossible to block the FITC signal from the
TRITC channel with your filters.  Our solution (which is
not possible on your machine) is to recommend the
combination of Cy3/Cy5 for double labelling, as Cy5 is
excited at 633 (from our HeNe laser) and there is no bleed
through from FITC that high up.  Another solution is to
collect data in both channels with 488 excitation, and then
subtract out the signal that appears in the TRITC channel.

I have never done an excitation spectrum on FITC, but I
would not be surprised if there was significant excitation
at 515.
----------------------------------------------------------
Herbert M. Geller, Ph.D.
Professor
Department of Pharmacology           voice -  908-235-4084
Robert Wood Johnson Medical School     fax -  908-235-4073
Piscataway, New Jersey 08854   Internet - [log in to unmask]
www: http//www2.umdnj.edu/~geller/lab/