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We ran a mixture of beads of different fluorescence intensities and found
different ratio's of fluorescence intensity depending upon the voltage
setting for the PM - tube. At higher voltages the ratio's "contracted".
The cause for this phenomenon is that at higher voltages the dynodes in the
PM - tube are more saturated. This can be demonstrated by a stepwise
increase in voltage over a PM tube and recording its output current. You
will find an S - shaped response to linear increments in voltage.
The correct procedure for calibrating a flow cytometer (or any other
instrument using PM tubes to monitor photon intensity) is to keep the
voltage of the PM tube constant, and to use a fluorescent standard (eg. a
fluorescent latex bead) to monitor the "output of today". Thus, data
recorded on different days (with different instrument adjustments) can be
compared by taking the ratio in fluorescence intensity of sample vs
standard. In this setup the degree of dynode saturation is constant and all
data can be readily compared.
Good luck !
Martin Poot
Molecular Probes, Inc.
P.O. Box 22010
Eugene OR 97402
martin@probes.mhs.compuserve.com
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