Just to add meine 2 Pfennige Whilst spectral overlap may be constant, one of the problems of compensation that it is indeed not a fixed value but depends on a number of factors, the dye characteristics and the response characteristics of the detectors. The latter is a combination of the spectral response mainly determined by the filter characteristics (which are quite different between different instruments even of similar models) and the amplification settings. For the simple case of the 'bleed' of FITC into the PE channel the value is constant for a fixed set-up of both detectors. Below this is described in a hypothetical example The signal received by detector FL1 is 1000 photons which the detector converts into 1000mV. Due to the FITC emission and the filter combination the FL2 receptor will receive 100 photons thus 100mV that can be compensated by setting fl2=fl2-10%fl1. A cell that sends 10,000 photos into FL1 (10,000mV) will therefore send 1000 photons into FL2 (1000mV) which is again compensated correctly with 10% as above. However, if one changes PMT voltages or amplification factors things change. If for example the FL2 amplification is doubled, the overlap of 100 photons will now generate 200mV, thus the 10% compensation is insufficient or it is to high if FL2 amplification is reduced. The inverse is true if only FL1 amplification is doubled. Whilst the 1000 photons will now generate 2000mV and the 100 photons in FL2 will still only generate 100mV thus a 10% compensation would lead to a -100mV signal. In case of a very bright green fluorescence one might set FL1 amplification low to get the signal in scale. If full scale would be 10,000 mV and 100,000 photons to be detected the amplification factor would have to be 1/10. At a 10% photon bleed this would send 10,000 photons in FL2, thus 100% compensation would be required. With the variation between instruments it is not possible to transferee settings from one instrument to the other. A point to remember here is also that filter characteristics are depending on the angle at which the photons pass the filter / mirror, a trick usually used to fine-tune optical filters. Hope this helps to clarify bits of the discussion Gerhard PS And if I remember right, the spectrum of a dye is not a constant...
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