(Go back one page, or back to the Introduction).

The goal of compensation is to remove the spillover fluorescence of a particular
probe from the "wrong" channel. I.e., fluorescein fluorescence
is primarily green, which is measured in the FL1 (FITC) channel. But fluorescein
also has a significant yellow component to the fluorescence, which appears
in the FL2 (PE) channel. One of the properties of fluorescence is that the
ratio of these two measured values, the fluorescein fluorescences in FL1
and in FL2 channels (green/yellow), is always the same (at any given PMT
voltage, filter set, and instrument sensitivity). In an ideal world, we
can exactly determine the yellow fluorescein fluorescence (in the PE channel)
based on the measurement of the green fluorescence (in the FITC channel)--and,
in an ideal world, we could exactly correct ("compensate") for
this yellow fluorescence and not worry about the contribution of FITC to
the PE channel. The result is that the output of the compensated PE channel
will represent only the "true" (yellow) fluorescence arising from
PE itself.

Mathematically, compensation from FITC to PE simply subtracts a fraction
of the FITC signal from the PE signal: the more FL1 (green fluorescein fluorescence)
there is, the more spillover into FL2 (yellow fluorescein fluorescence)
there will be (i.e., as stated above, the green/yellow ratio does not change).
As an example, if the amount of yellow fluorescein signal in the FL2 channel
is 15% of the green fluorescein signal in the FL1 channel (i.e., "15%
compensation"), then we can exactly determine the "true"
(or "pure") PE fluorescence of a cell, even in the presence of
FITC fluorescence, as:

This process is identical for correcting for PE fluorescence appearing
in the FITC channel. For instance, if the amount of (green) PE signal in
the fluorescein channel is 2% of the (yellow) signal in the PE channel (i.e.,
"2% compensation"), then we can exactly determine the true FITC
fluorescence of a cell as:

These equations hold true even if a cell has both FITC and PE fluorescence
(the machine always does these compensations independently and simultaneously;
i.e., it does not use the output compensated FITC value as an input to the
PE compensation).

If you would like a detailed example of this process, then click here.
The example is not part of this informal discussion; you will have to navigate
back to this page manually. Please note that the equations given on this
page are only correct for doing **one** compensation at a time--because
of this, the compensation coefficients given on this page (as well as the
equations) are somewhat different than those in the detailed example. To
simultaneously compensate two or more colors, compensation requires the
generalized solution derived with matrix algebra, described elsewhere.

Otherwise, Go on.