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As the number of available probes and detectors continues to increase, the logistic demands on optimized panel development become too high to guarantee success by empirical methods. In addition, the SSM is critical information for predicting immunofluorescence panel performance. Individual SSM values perform as predicted by theory i.e., they reflect the Poisson-based error in fluorescence quantification. As we show here, the SSM is a useful indicator of instrument performance, and can be used to evaluate a single instrument over time, as well as compare different instruments.
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Compared to the compensation/spectral overlap matrix 1, the SSM provides independent information about measurement performance. The aggregation of all spillover spreading values together in a matrix is termed the spillover spreading matrix (SSM). Here we derive an intrinsic measure of spillover spreading. We hypothesized that quantifying the degree of spillover spreading between every pair of measurements will be valuable for comparing instruments ( 2) and for guiding immunophenotyping panel design ( 3– 5). This error is proportional to square-root of the intensity of the signal this leads to a non-linear (but straight-lined on a log-log plot) “spreading” of properly compensated data ( 1). In current instruments, a large source of error is the Poisson error associated with photon detection (“counting error”).
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The more signal from dyes with fluorescences that spill into a detector, the more the sensitivity in that detector will be diminished. A major impact of fluorescence spillover (which occurs when fluorescent probes emit light into multiple detectors) is a reduction in sensitivity (defined as the lowest signal detectable above all sources of background).