Dear Mario--

Mario M. Moronne wrote:
> If you had fully packed
> antibody averaging four fluors per molecule, I get an estimate of about
> 0.11 OD absorbance through half a micron assuming an extinction coefficient
> of 80,000 for the dye. That means about a 25% drop in power through the
> volume or an average illumination intensity of about 87%. That's not a big
> effect by biological standards. Further, it needn't be said that fully
> packed antibody to half a micron will never occur in a real sample. The
> case where inner filter effects would come up is with small molecule
> labeling, where you can get a very high local concentrations. One case
> might be with DNA stains in the nucleus.

If you have a fairly uniform distribution of dye (and thus of
absorbance) throughout the tissue, the absorbance of the tissue above
the region of interest will decrease the intensity of light at the level
of the ROI.  Thus inner-filter effects are perhaps not so far-fetched.
We think we've seen inner filter effects in cases of strong labeling and
thick objects.

> The interaction of dye molecules at high concentration has also been
> mentioned as a contributor to non-linear effects. There are cases where
> this can happen but it requires that fluors reach distances closer than say
> about 100 angstroms so that energy transfer can occur between dyes or that
> the dyes can interact directly forming dimers such as the case of
> rhodamine, which gives rise to quenching. These conditions are not likely
> to occur when using macromolecular probes such as antibodies or their like
> since the dye concentrations never become significant in this context. One
> mg/ml antibody will typically have a fluor concentration of 30 uM. Usually,
> self quenching of fluors isn't seen until you get to concentrations greater
> than 10 mM.

Again, I think that this can occur in "real life".  For instance, Bodipy
(the original green stuff) has a wonderful excitation-emission spectrum
and based on it you'd never expect problems with inappropriate
fluorescence.  Nevertheless, when you single-stain tissue with it, in
areas of strong labeling you can obtain distinct, surprisingly bright
red emission using green excitation (Hg burner) or the 568 nm line of a
Kr-Ar laser.  Someone once told me that Dick Haugland explained this as
the result of dimer formation at high concentrations.

My guess is that when antibodies get packed onto tissue, that the
effective local concentrations are quite high.

Best regards--

Martin
--
Martin Wessendorf, Ph.D.
Associate Professor, Dept Cell Biology and Neuroanatomy
University of Minnesota
Minneapolis, MN  55455
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