The Total Spreading Matrix (TSM) is a Spillover Spreading Matrix (SSM) without normalization to the fluorescent intensity of the probe. This makes it possible to evaluate the true spreading in a given panel, based on the signal intensity of the different marker/fluorochrome combinations in each channel.
TSMs are calculated in FlowJo 10 when compensation is calculated from single stain controls using traditional gated populations for positive and negative subsets in the compensation wizard setup. A TSM will not be calculated when using the AutoSpill function.
Calculating an TSM
To generate a Total Spreading Matrix in FlowJo visit the compensation editor, select any compensation matrix that was calculated in FlowJo using traditional gated positive and negative subsets from single color controls, and then click the “SSM” button. Select "Display Total Spread Matrix" to view the TSM in the FlowJo. Select "Export Total Spread Matrix" to save underlying TSM values as a CSV file.
Please note that TSMs will not be available for Acquisition-defined matrices or compensation matrices calculated in FlowJo using the AutoSpill function, as the calculation depends on statistics from the single color control gated positive and negative subsets.
Why Calculate TSM?
- Panel optimization – Immunofluorescence panel design becomes exponentially more complex as researchers utilize increasingly more channels of detection in their experiments. The TSM calculation helps point the way to maximize useful signal.
Webinar on using TSM as a tool for use in panel design and evaluating panel performance:
For additional guidance on the use the Total Spread Matrix as a tool for panel design, check out this webinar: https://www.youtube.com/watch?v=40SuNMGZfpY