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Due to their optical design, EPICS instruments can introduce an artifact
that looks like increased FITC background when using a dual laser UV/488
setup to measure Hoechst and FITC. Because the EPICS does not spatially
separate the beams the FITC PMT (which can be the same one used to measure
Hoechst) sees the often much brighter Hoechst emission. This can swamp the
analog electronics to the point that the DC level cannot be restored in
time for the FITC measurement to be made. The effect is exagerrated
because the FITC measurement is often processed through (ugh!!) an
analog log amplifier.
One solution is to always make sure that the 488 beam is thetop laser
and to decrease the UV excitation (often with ND filters) such that
the Hoechst emission does not saturate the FITC analog channel.
Optical designs which spatially resolve the laser-stream intersection
points should not suffer from this problem.
Gary Durack
University of Illinois Biotechnology Center
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