![]() So, if anyone has ever positioned the same parameter on both axis of a dot plot, you'll know that the graph that will apear is a perfect diagonal line (45 degrees, passing through ZERO). When this factor is set properly, even though the voltage pulse is different between A and H, they will have the same reported value (that will be used to represent this particular event in the dot plot). In BD instruments we call this Area Scaling. Digital instruments have a function that allows one to correlate A and H so that they present the same variation when PMT voltages are changed, so that they can be correlated one to the other. The basics of doublet discrimination is to detect disproportions between cell size vs. So, summarizing, we have H as the best correlation with highest signal (for example expression of a specific protein), W as the best correlation to cell size and A as the most completely and realistic cell scenario, cause it associates signal/expression correlated to cell size. It can be processed in up to three differente forms, two of them measured and one calculated: Height (H), cell's maximum signal, is directly impact by any changes in PMT voltage Width (W), which represents time duration of the signal, not impacted by PMT voltage, directly correlates with cell size Area (A), calculated from all H measurements during the time (W) the cell passes through the laser beam, variates with PMT voltage changes but not in the same magnitude that H (since W doesn't change when PMT voltage changes). This voltage pulse will be processed and defined by a value. There is a voltage pulse per cell for each parameter your cytometer detects. As Paolo explained a voltage pulse represents cell's signal when excited by the laser beam. However I do like a lot FSC-H vs FSC-W and SSC-H vs. There are different ways of discriminating doublets. ![]()
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