Although we haven't done so much in the way of confocal work yet, we have
worked on this problem for many years with standars fluorescence
microscopy. Although there is a slight difference in emission
characterisitics of FITC and BODIPY, in practice it doesn't really make
that much difference. Texas Red with its longer emission spectrum than
rhodamine helps a lot, but in the end you need really narrow band
barrier filters. We had some made from Omega a couple of years ago. They
have high transmittance but very narrow band pass. This means that you lose
a lot in brightness, but you gain the specificity. The loss in
brightness is not usually a problem in confocal work. Even for
conventional fluorescence microscopy we get over this probelm by using
sensitive video cameras and integrated fram averaging.
 
The filters seem to be  difficult to make so that they work in a real life
application. Some that we tried had excellent filtering properties but had
terrible optical performance. We ended up with one that really clearly
selects FITC from any of the red fluorophores - rhodamine, DiI, Texas Red,
Cy3 etc: it has a band pass of 520-550 nm centred on 535nm. It was made for
us by Omega. Some other companies have filters with similar specifications
on paper, but they don't seem to have such a sharp cutoff. You also need to
fiddle with the excitation filters as well, so that a highly discriminating
set of filters will have narrow band pass filters on both the excitation
and emission side. Few of the microscope companies seem to be alert to
this. However, Olympus now has an arrangement with Chroma (who also make
excellent filters) so that they will make custom blocks up for the new BX /
IX range of micrscopes. We are just about to try Chroma for a set...
 
Hope that helps..
 
IAN
Ian Gibbins
Department of Anatomy and Histology
Flinders University of South Australia
Phone:  +61-8-2045271
FAX:    +61-8-2770085