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Hi Suzana,

In my observations, this is happening quite often;  I've seen this
effect using a wide field, the plane confocal imaging and, especially,
using multi-photon excitation of dyes.
I believe this natural expansion of the DAPI emission spectra toward
long wave range is always occurred for UV-shined DAPI,  maybe, due to
some changes of whole DAPI-DNA complex or DAPI molecules alone.
To prevent this, I always advise our users to follow rule of thumb: to
scan (or take an image) of the fluorescent marker with a longest
excitation maxima first and,  then, take images of dyes with shorter
excitation wave length.
So, DAPI is last in line.

Suzana Glavas wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Perhaps somebody can help me make sense of this.   We have a GFP tagged
> protein expressed in C. elegans embryos.  Since the GFP signal is not
> very
> strong, we use an anti-GFP antibody and a FITC tagged secondary to better
> see this particular protein.  When we visualize the FITC stain, we see
> nice
> membrane localized staining with very minimal background.  We then switch
> over to DAPI to look at the nuclear staining.  When we switch back to
> FITC,
> all of the nuclei can now be seen in the FITC channel.  We are
> visualizing
> with an epifluorescence system prior to doing confocal.  Any ideas what
> might be happening?
>
> Suzana Glavas
>
>

--
Edward Monosov, Ph.D.
Director , Cell Imaging & Histology
The Burnham Institute
10901 N. Torrey Pines Rd,
La Jolla, CA 92037
Ph:    (858)646-3100 ext. 3206
Fax: (858)646-3196
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