"Negative fluorescence"?

Recently, I was queried by a user who complained about the new data  
transformations (Biexponential, Logicle, Hyperlog, etc.), which show  
negative values.  The user complained that negative fluorescence is  
not biologically meaningful.

Indeed, the user is correct.  There is no meaning to negative  
fluorescence; there is no such thing.  However, what we are  
displaying on the graphs is no longer fluorescence, it is a corrected  
measurement derived from fluorescence.	The corrected measurement has  
been manipulated by both the instrument firmware, and possibly	
software (in the case of compensation), before it is displayed.

When the fluorescence is detected off the PMT, the voltage  
measurement has "baseline" subtracted from it.	Baseline is measured  
during the time between pulses.  However, there is an error in the  
measurement of both the fluorescence as well as the baseline.  So,  
for "negative" cells (cells with little or no fluorescence), there's  
a chance that the measurement for the cell will be less than the  
measurement for the baseline, because of the errors involved.  After  
subtraction, the measurement is less than zero.

Likewise, for compensation, additional measurement errors from all of  
the compensation channels are aggregated into the final computed  
value; thus, frequently this value is less than zero.

Look at it another way:  there is a distribution of measurements  
around the true value; that distribution is dictated by the error in  
the measurement (i.e., the SD).  If the true fluorescence is nearly  
zero, and the error is fairly large, then of course we expect the  
distribution to rise to the positive values -- with 95% of the values  
being within a few SD of the "true" value.  But measurement errors  
are always symmetrically distributed--so the distribution has to  
reach into the negative values just as far.  It would be incorrect to  
set these negative values to zero, because they you would  
systematically increase the mean of the distribution (you are always  
increasing negative values, but you are not decreasing any other  
values).

But the bottom line is that we are not displaying measured  
fluorescence values -- those of course are always positive.  We are  
displaying corrected measurements.  Notably, the corrected  
measurements are still linearly related to the fluorescence of the  
original event.

mr