As you build a panel (or FluoroFinder's Intellipanel builds one for you), FluoroFinder calculates "Spillover" and generates a "Spillover" matrix presented to you on the "Products" and "Summary" pages of our panel builder. But what is this data, how does one interpret it, and what does it mean for our experiment?
What data is presented in FluoroFinder's "Spillover Matrix"
What data is presented in FluoroFinder's "Spillover Matrix"
As your fluorophore choices are made, FluoroFinder calculates what percentage of the target channel for each fluorophore is covered by your selection. For example, in the Spillover Matrix below, the red rectangle highlights that emission of the selected fluorophore, Brilliant Violet 510, will cover 88.7% of the "area" of the 525/50 bandpass filter (its target "channel") on our cytometer. We will call this value "filter coverage":
Filter coverage of Brilliant Violet 510 in the 525/50nm filter
Each of the numbers on the diagonal of the "Spillover Matrix" represent the "filter coverage" for a fluorophore in its primary detection channel on your selected cytometer (see red rectangles in image below):
Filter coverage is represented for each fluorophore on the diagonal of the Spillover Matrix
The remaining values in a ROW represent the "Normalized Spillover" of a fluorophore into the primary detection channels of each OTHER fluorophore in the designed panel. This is calculated using the following formula:
(Fluorophore coverage in off-target filter)/(Filter coverage in primary filter)
The example below highlights the "Normalized Spillover OUT" values of Brilliant Violet 510
Normalized Spillover OUT values of Brilliant Violet 510 into other used channels
Shading of the grid within the Similarity Matrix indicates areas of significant "spillover". The shade of red coloration indicates the severity of "spillover", and the degree to which that "spillover" may contribute to compensation and complexity issues in the final panel.
What does this data mean for my experiment?
What does this data mean for my experiment?
While there is a wealth of data represented in the "Spillover Matrix", and a full analysis of its importance to your experiment is a topic for discussion with your Flow Core Manager or local Flow Cytometry expert, there are a couple general statements we can make about our "Spillover Matrix":
Filter Coverage - this metric is a good guide for determining "is the selected fluorophore appropriate for the detection channel I want to view it in?". High filter coverage indicates that a fluorophore will likely be easy to visualize in the channel.
High Spillover - areas of high spillover (pink/red coloration) in the matrix indicate potential areas that may make resolving your target populations difficult. When looking at areas of high spillover, consider if the fluorophores selected are excited by different lasers, located on different cells, or if perhaps different fluorophores need to be selected to minimize spillover.