Could someone provide a more detailed explanation as to how autoflourescence values are handled in the spillover and compensation functions in the flowCore package? I can't figure out how to view the function's code...
I know that the autoflourescence/baseline data can be specified in the spillover function with a blank/unstained control sample, but I don't know what happens with it next. Is autofluoresence calculated from each channel and linearly subtracted from the data before the compensation is applied? Is each channel's background incorporated into the spillover calculation as seems to be described in the 1993 paper that the function references? Is this considered a non-linear, affine transformation and therefore different from simply subtracting median AutoFl values from the uncompensated data? If my workflow typically calculates median fluorescence from an unstained control and linearly subtracts that from my sample flowSet prior to calculation of the spillover matrix and compensation, am I correcting for autofluorescence twice?
any help, guidance, or suggestion is welcome.