SpectralFXTM is a proprietary version of spectral unmixing available on the BD FACSDiscoverTM S8 that can also be enabled in FlowJo.
In spectral cytometry there are many ways to ‘unmix’ the signal produced by one fluorochrome, measured using all detectors, from other fluorochromes. The most common method is Ordinary Least Squares (OLS) unmixing, which is used by default in FlowJo when the spectral box is checked in the compensation wizard for any set of data with more detectors than fluorochromes.
The OLS solution assumes that all detector signals are equally noisy. However, we know that this is not true, because detectors can have varying background noise levels, and fluorescence measurements are Poissonian; the measurement gets noisier as the signal gets brighter. SpectralFXTM system-aware unmixing, available for spectral data acquired and unmixed on the BD FACSDiscoverTM S8, uses information about the instrument’s noise levels to help mitigate spread in spectral data.
As of FlowJo version 10.10 there is an additional unmixing option called SpectralFX for data from specific BD cytometers that include keywords which provide information about the relative noisiness of the detectors. Unmixed recordings from the BD FACSDiscoverTM S8 contain hard-coded unmixed FCS parameters that contain the unmixed data generated at the time of acquisition (located between scatter and raw fluorescence parameters in the parameter selection menu), as well as raw fluorescence detector data which may be used for re-unmixing during data analysis in FlowJo.
If SpectralFXTM unmixing was performed on the instrument, the acquisition spillover matrix and necessary FCS keywords to recreate SpectralFXTM unmixing will be available in the FCS file. Results generated via unmixing in FlowJo will appear as compensated parameters with a “(SW-unmix)” suffix (located at the end of the parameter selection menu). This unmixing technique may be applied using the acquisition matrix or any other spectral spillover matrix (e.g., generated using single-stain controls through FlowJo’s compensation wizard, or edited from the original acquisition matrix). This means that SpectralFXTM unmixing may be applied to data using a different spillover matrix than was used at the time of acquisition, which may be helpful for troubleshooting and correcting unmixing errors. To apply SpectralFXTM unmixing in FlowJo, right-click on a file or group of files and choose SpectralFX from the menu:
FlowJo will then apply SpectralFXTM, refreshing any open plots, and taking a short amount of time to complete. The effect may be subtle for data sets that have been well compensated, but we will generally expect to see some reduction in spreading.
With SpectralFX TM Without SpectralFXTM
To remove the SpectralFXTM effect, simply return to the dropdown menu and select the next option down “Apply OLS”.