Dear all,

        We are trying to study the uptake, distribution and localization of
porphyrin conjugates in MCF-7 cells. So far, the specimens analyzed have
shown some problems which I describe below:
a) When testing the porphyrin conjugate by itself in DMSO, the emission was
the expected in the 600 nm range. However, when futher diluted in aqueous
solution, there seemed to be a shift in the emission that could not be
detected with the filters available in our scope. We are working on the
fluorescence spectra to examine possible differences between the porphyrin
in DMSO and aqueous solutions. Has someone observed a phenomenon or this sort?
b) The fluorescence observed in cell preps is weak (we have an Ar laser and
use the 514 nm line for better excitation). We may still be missing the
peak of emission, but total fluorescence is also low (maybe due to low
uptake).
c) Is quenching and/or photobleaching an issue for porphyrins? (our chemist
says he keeps them on the bench for a long time with no problem). Should we
include antifade agents?
d) The whole prep looks blurry. Is there a possibility of leakage of
porphyrin back out of the cells into the mounting medium? Which fixation
methods do you suggest? We have used formaldehyde/glutaraldehyde plus
permeabilization with Triton-X-100.
e) To have a "gross" positive control for visualizing the porphyrin, we
would like to induce a massive reversible uptake of porphyrin by the cells,
even if it is non-specific. Is there any drug to do this?

        Sorry for this lengthy message, and we appreciate all your suggestions and
comments.

Susana and David

Vitamin D Lab
Boston University School of Medicine
Boston, MA 02118
Phone: (617) 638 8899