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The biggest problem will be that, although there will be a scatter pulse, the
fluorescence signals will not be pulses, but rather DC signals due to the dye in
the sample stream. When a bead passes through the laser intersection, there will
be a dip in dye concentration, yielding a dip, rather than a pulse, in the DC
signal on the fluorescence channel. Most commercial electronics will not handle
this well.
Along with David Parks and Lisa Peterson, we presented a method for calibrating
free dye in a sample stream at SAC X in 1984. We built an integrator which was
put between the preamp and amplifier on the fluorescence channel. The integrator
would integrate the DC fluorescence for a fixed time, then output two pulses,
one on the fluorescence channel whose height was proportional to the integrated
signal, and one on the scatter channel of fixed height to trigger the cytometer.
This was compared to a bead signal to get a measure of the dye/bead signal
ratio.
We used this method to calibrate antibody bound FITC in terms a bead pulse
height. (Note that the absorbance F/P ratio is not a quantitative predictor of
the actual fluorescence output). This allowed us to find the antigen density of
particular antibodies on cell surfaces.
-Marty Bigos
Stanford Shared FACS Facility
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