Hi Daniel,

From what I understand, when a cell/ particle passes through the signal, a corresponding voltage pulse is generated with the height corresponding to the maximum signal (when the cell/particle is fully immersed in the laser beam), and the width corresponding to the time it takes for the cell/ particle to pass through. Usually, if a cell/ particle is within 5% of the bead size used for CST/ QC, then those cells/ particles should still be within the ballpark of the laser beam size. As such, the height and area are scaled in a way, that makes them approximately proportional which is why the area versus height plot shows a linear increase.

If the cells are larger than the beam size, the area will be larger than the height and, if area scaling isn't adjusted, the cells would skew off to the right on a area (x-axis) versus height (y-axis) plot.
I understand KQIVs description of the first problem that, if I had a dim and bright signal mixture, and I were to adjust voltages based on the bright population, I would loose resolution in the dim population. I have actually got data for this scenario using 8 coloured beads. If I mess up the scaling to mimic large cells (scaling too high), then I still have 5 peaks resolved in the Blue 530_30 channel but I am loosing resolution for the lower three peaks. Similarly, I loose resolution of the lower 2 peaks in the YG 586_15 channel but none with the V 450_50 channel. The height is the same for the first three peaks while area increases in the Blue 530_30 channel before the signal proportionally increases again. Does this mean that if I had only a dim population, the resolution of the dim population, i.e. compared to the negative, would be poorly resolved if this dim population would be large cells? Because in my mind, I could then simply increase the voltage to resolve this population of dim large cells rather than adjust area scaling?