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Note that there is a significant spectral shift of Hoechst emission near the
saturation concentration in binding to nuclear DNA. We, and James Watson,
saw the change in spectra when Hoechst reaches maximum binding. Therefore,
Hoechst bound in saturating levels to DNA has different fluorescence
emission spectral properties as compared to free dye. This points out the
problem of using beads with dye to set compensation electronics. Chromophores
coupled to beads (inside or outside) do not provide the proper controls to
set compensation properly. It is crucial to run each color alone, with the
cells and reagents used in the final multicolor experiment, to correctly set
compensation.
If the cells are to be fixed, why not use a red emitting chromophore for the
DNA rather than blue? Keep in mind that all fluorescence spectra have a red
tail but are sharp on the blue side. The brightest stain, e.g. DNA, should
always be to the red part of the spectrum while the dimmer stain
(immunofluorescence) should be to the green or blue to reduce compensation.
Otherwise you are fighting a loosing battle.
Without the constraint of using Hoechst, Howard would have suggested to use a
HeNe laser rather than a UV laser. Clearly this is the right way to go with
fixed cells.
Mike Loken
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