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.
Thanks,
Thank you for your help! That being the case, I'm confused as to why the mean/median for my unstained controls is practically the same in both the uncompensated and compensated data. If the blank mean/median is rolled into the compensation matrix, compensation should transform the mean/median to zero, right? Could you shed light on this?
> namePattern <- "FSC.A|SSC.A|V1.A|Y2.A" ## set the desired channels, in order
> comp.1 <- spillover(x=fs.colorcomp.1, unstained="000.001", fsc="FSC.A", ssc="SSC.A",
+ patt=namePattern, method="mean")
> fs.comp.1 <- compensate(fs.clean.1, comp.1)
> summary(fs.clean.1[[3]])HDR.T FSC.A FSC.H SSC.A SSC.H V1.A V1.H Y2.A Y2.H
Min. 952 28010 11050 772.4 278.1 -80.56 -1.831 -101.700 -1.492
1st Qu. 59620 49610 21270 9000.0 5504.0 11.09 55.990 -11.490 47.620
Median 127600 63840 27550 11750.0 7093.0 31.57 72.020 9.788 62.370
Mean 128900 67320 28840 12530.0 7455.0 31.59 69.630 10.120 62.320
3rd Qu. 196700 82090 35290 15470.0 9096.0 51.38 84.380 31.820 77.140
Max. 262100 136000 64970 24930.0 16770.0 708.20 544.400 280.800 198.700
> summary(fs.comp.1[[3]])
HDR.T FSC.A FSC.H SSC.A SSC.H V1.A V1.H Y2.A Y2.H
Min. 952 28010 11050 772.4 278.1 -80.51 -1.831 -101.700 -1.492
1st Qu. 59620 49610 21270 9000.0 5504.0 11.05 55.990 -11.490 47.620
Median 127600 63840 27550 11750.0 7093.0 31.55 72.020 9.788 62.370
Mean 128900 67320 28840 12530.0 7455.0 31.58 69.630 10.120 62.320
3rd Qu. 196700 82090 35290 15470.0 9096.0 51.37 84.380 31.820 77.140
Max. 262100 136000 64970 24930.0 16770.0 707.90 544.400 280.800 198.700
>