Colleagues, Thanks to all who answered my follow up question to the need to compensate APC from Pe-Cy5. The initial replys that suggested there might be a persistence of signals generated by the blue beam that then entered the FL4 channel although debunked by Dr. Shapiro, fluorescence signals lasting on the order of nanoseconds and the travel time between the two beams lasting on the order of microseconds, nonetheless gave me food for thought and were much appreciated. Tom Frey's explanation is quite clear. I think I was misled by the graphic used in class which illustrated signals from both beams being overlayed during the Time-Delay calibration on the Calibur. If I now understand Tom correctly the signals generated from the two beams are not electronically processed simultaneously - such would in fact prevent the instrument from distinguishing red excitation from blue excitation - they are compared simultaneously to permit analog subtraction for interbeam compensation. Gene Pizzo/UCONN Health
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