Gene Pizzo asked: >>>>I'll ask the question again...it hasn't been answered here yet, twice. >>>>It wasn't answered in BD class when I asked it. >>>>It wasn't answered by the service engineers I've asked. >>>>Maybe no one has an answer. Well, I feel kind of compelled to put in my two cents now! Unfortunately, I also feel an urge to the longwinded today - you are warned. >>>>We all understand that the Cy5 of the PE-Cy5 tandem can be excited >>>>by the red laser, that's the easy part. But if the signal generated >>>>from the two beams is separated in both space and time why should >>>>there be a compensation necessity? Compensation is used to remove the true fluorescence of a dye from channels that we don't want it to show up in, so that we can then assign one antibody to one channel. (For example fluorescein REALLY emits in FL2 at 585 nm, we just comp it away) PerCP and PE/Cy5 are each excited by the red and the blue lasers and emit long wavelength red. The separation of the beams is what allows us to tell whether the emission is from red excition or from blue excitation. Since both PerCP and PE/Cy5 are "FL3 dyes" we want to subtract their red excited fluorescence from the FL4 channel. >>>>Furthermore, why on the FACSCalibur is it necessary to align the two >>>>beams in time when on the FACStar Plus the electronic separation of >>>>the two beams in time is considered a necessity? For those you who haven't used a four color Calibur, I think we're discussing the Time-Delay Calibration function that is part of the Calibur. This function does NOT align the beams in time. It (more or less) matches the timing between pulses from the two beams in the electronics to the travel time between the two beam intercepts. It is intended to allow for changes in the particle velocity in the sample stream. The pulse from the red beam is delayed to arrive simultaneous with the pulses from the blue beam. The delayed signal is used for interbeam compensation. The Calibur does this compensation in analog, the pulses are subtracted over the entire pulse waveform. (I don't want to get into an argument about whether this is "best" - there are good reasons to do things this way in a 10-bit system (and no I don't know where bit came from)) For analog compensation between beams the pulses from the two sources need to be properly aligned - ie the peaks need to occur at the same time. If, during time delay calibration, the system senses a misalignment of the pulse originating from the blue laser (forward scatter in this case) and that from the red laser (FL4 from the recommended APC beads) then the electronics are adjusted so that the pulse alignment is corrected so that accurate subtraction can occur. I hope this goes some distance to answering the original question without inducing too many blank stares. I am sure, as always, the group will clarify any points they feel are incorrect or too vague.
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