Simon Taylor writes:
> To all confocal users
>
> Our institute has recently purchased an Optiscan Personal Confocal System
> model F900e.  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.
>
> I would appreciate any feedback on the above problems.  Thanks.

Dear Simon--

Get an additional laser (or additional laser line) into your system.  It needs
to have a wavelength long enough that it won't excite FITC (i.e. longer than
about 535 nm).  A 543 nm HeNe should be suitable; the 568 nm line of a Kr/Ar-ion
laser is suitable.  With that, you can excite rhodamine without exciting FITC,
and your efficiency of excitation will probably be higher, too.

Good luck!

Martin Wessendorf PhD, Associate Professor           [log in to unmask]
Dept Cell Biology and Neuroanatomy                      (612) 624 2991 (voice)
Confocal Microscopy Facility                              (612) 624 8118 (FAX)
University of Minnesota
4-144 Jackson Hall
321 Church St. SE
Minneapolis, MN  55455