Autofluorescence
(Go back one page to generalized compensation, or
back to the introduction)
Autofluorescence throws a small kink into
compensation (as it does into many things), but, as it turns out, does not
change the ability to deconvolute spillovers.
Cellular autofluorescence is present in all channels
to varying extents, and provides a background (that varies from cell to cell).
There are two ways to deal with autofluorescence.
One way is to devote a single channel to measure autofluorescence.
Because the autofluorescence spectrum of cells is
generally very similar, we can just treat autofluorescence
as one more type of fluorescent molecule. Now, by compensation, we can actually
correct for the contribution of autofluorescence to
all channels except one. This process can significantly enhance sensitivity for
detection of low-density antigens. Autofluorescence
compensation is more fully described in published references (see Roederer
& Murphy, "Cell-by-cell autofluorescence
correction for low signal-to-noise systems: application to EGF endocytosis by
3T3 fibroblasts," 1986, Cytometry 7:558; and Albert, Parks, & Herzenberg, "A single laser method for subtraction of
cell autofluorescence in flow cytometry," 1987, Cytometry
8:114).
The second way is to simply ignore it--apply the matrix algebra described
previously still works to make the channels independent (i.e., with only autofluorescence and the specific fluorescent molecule of
interest contributing to the channel). The compensated fluorescence value is proportional
to the concentration of the fluor, which is what we
want.
Simple compensation in the presence of autofluorescence,
therefore, works just fine: the resulting values are independent of the
presence of other reagents, and are proportional to the amounts of the
fluorescent molecules present. That's all we really need for flow cytometry.
Note, however, that if the autofluorescence of the
stained cells in the compensation sample is different than that of the
unstained cells in the compensation sample, then the computed compensation will
be incorrect! Thus, you could not use FITC CD14 (staining highly autofluorescent monocytes) to compensate against unstained
lymphocytes (which have low autofluorescence).
Go on to the requirements for proper compensation.