The SSM calculation helps point the way to maximize useful signal.Ĭomparison between different instrument configurations – The measurement gives a PI or SRL manager the ability to quantifiably contrast between different cytometers and/or alternate arrangements of a given cytometer. Panel optimization – Immunofluorescence panel design becomes exponentially more complex as researchers utilize increasingly more channels of detection in their experiments. Quality control of instrument sensitivity – Since signal spreading directly effects channel sensitivity and results between similarly configured machines are readily comparable, the SSM can be used as a benchmark of configuration and performance quality control. The values in these matrices are directly comparable, though they only describe one part in a complex system which contributes to the quality of a flow panel. The values within this matrix are not a percentage, but represent a standard deviation of spillover, AKA signal spread. These values can be calculated for each detector staining pair, resulting in the spillover spreading matrix. F is fluorescence in units of intensity.Sigma is standard deviation (adjusted to use 84th and 50th percentile).S stands for ‘Stained’, meaning the FL2+ population in this example.R stands for ‘Reference’, meaning the FL2- population in this example.The spillover spreading for a given detector/spillover-color pair can be calculated empirically by taking the square root of differences in squared robust standard deviations of measured fluorescence intensity from unstained negative and singly stained positive control populations: Spillover spreading is construed as the impediment of signal detectable above background, and is intrinsic to the configuration of a given instrument.

To generate a spillover spreading matrix in FlowJo visit the compensation editor, select the matrix of interest and click the “SSM” button there:

This tool works for matrices calculated within FlowJo’s Compensation Wizard only, it isn’t possible to calculate SSM for acquisition defined compensation matrices calculated beforehand. The Spillover Spreading Matrix (SSM) is available from the Compensation Editor.

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