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If you look at the excitation spectrum of PE you will see that out at the
dye laser wavelength range PE does exhibit some excitation. So seeing a
signal proportional to PE in the TR detector is to be expected.
There are several things that can be done to minimize this.
1) Adjust your dye laser wavelength to a slightly longer value. This will
minimize excitation of PE. It will also result in a somewhat less optimal
excitation of TR. We have used 600nm here with good results. You will also
need to check to make sure no dye laser signal is being scattered into the
TR measurement channel; we have seen in the past TR filters that were less
than optimal in blocking the dye laser.
2) Select your reagents so that the brightest reagents are on TR and duller
ones are on PE. This will help minimize the problem.
3) If you are not sorting, run compensation controls and do software
compensation between PE and TR. If you are sorting, badger the
commercial companies to put some development effort into cross-laser
compensation.
Hope this helps.
-Marty Bigos
Stanford Shared FACS Facility
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