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It occurs to me that the following alternative strategy might work. A
fluorescein solution can be made with known molecules per nanoliter based on
the weight of pure dry solid dissolved. Using this to suspend nonfluorescent
beads gives a signal since the bead triggers the forward scatter threshold,
and the fluorescence of the core stream volume outside the bead is then
reported by the cytometer. By using two beads of substantially different
volumes (e.g. 2 micron vs. 5 micron diameter), the difference in fluorescence
signal can be ascribed to the difference in bead volumes (the larger bead
giving the smaller signal since it displaces more of the core stream). The
difference in volume can be expressed as a known number of fluorescein
molecules; hence, this provides a MESF calibration of the instrument scale.
This strategy requires that the core stream diameter be constant, that is,
unaffected by either bead. Presumably, at very small core stream diameters,
the larger bead may produce a bulge/distortion in the core stream. This
artifact could be avoided by increasing core stream diameter (sample pressure
differential) until the fluorescence difference between the two beads becomes
constant. The optimal core stream diameter would be the smallest one which
satisfies this criterion since large core streams will be too bright to permit
accurate determination of the difference between beads.
Common cytometers (e.g. the FACScan) measure fluorescence peak height by
default. This is advantageous since, unlike the peak area, the peak height
should be unaffected by the difference in time that the different diameter
beads exceed threshold.
It seems to me that the best way to see if this works is to try comparing the
results to those with the FCSC beads.
Has anyone tried this or seen such a method published or proposed? Does
anyone see any pitfalls I've missed (short of getting actual data)?
--
/* - - - - - - - - - - - - - - - - - From - - - - - - - - - - - - - - - - - -
Eric Martz emartz@titan.ucc.umass.edu Professor of Immunology
Voice 413-545-2325, FAX 413-545-1578
Morrill IVN Rm 203, Dept Microbiol, Univ Mass, Amherst MA 01003 USA
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