Hi folks,

This subject has concerned me a lot, being from the flow cytometry
community and well versed in emission overlap problems.  We had lots of
problems until we figured out that we could get away with using the red
optics for a less significant label and the FITC and CY5 optics for the
important ones.  In our case we do our surface labelling with FITC and
CY5, and use phalloidin-RITC just to visualize the cells.

On another subject.  I want to do a 3D rebuild.  I can get the slices, put
them into a montage, but cannot figure out how to make NIH-Image do a 3D
image.  Can someone please talk me through this??

Thanks,

Deb Berglund
Montana State University

On Wed, 11 Dec 1996 [log in to unmask] wrote:

>
> There is a real problem emerging here that people on the list may like to
> comment on... As fluorophores, optical systems and detectors all become
> more efficient, getting complete discrimination between different
> fluorophores in multiple-labeled preparations is becoming more and more
> difficult with the current lot of labels. We recently got some very narrow
> band barrier filters made for our BioRad 1024 system, especially for the
> PMT detecting red fluorophores, so that there would be minimal fluorescein
> bleed through. Although in the end we would have been picking up just the
> very end of the emission spectrum of fluorescein, it still could be
> detected quite easily by the detectors set with relatively moderate gains.
> We also have seen this occur in our wide-field fluorescence microscopes.
>
> What can we do about it? The easiest option is to use fluorophores well