Spectral Unmixing

Spectral flow cytometry is used to sample more parameters per single cell or particle than traditional flow cytometry. In contrast to conventional flow cytometers, spectral flow analyzers and sorters substitute mirrors, optical filters, and photomultiplier tubes (PMTs) with a spectrograph and multichannel detector to use fluorescence or Raman spectroscopy.

With spectral cytometry instruments, a continuous, high resolution, optical spectrum is collected for each event in the sample. The spectrum is the sum of the spectra derived from all the dyes present on the event of interest. Spectral Unmixing is the process of transforming the data to determine the contribution of each dye to the total signal.

Spectral Unmixing with FCS Express

FCS Express fully supports creating spectral unmixing matrices from single stained controls.

Learn more with the FCS Express Spectral Unmixing Tutorial

To create an unmixing matrix in FCS Express simply use the Automatic Unmixing Setup wizard. The unmixing wizard functions similarly to the FCS Express compensation wizard making it easier than ever to move from standard flow cytometry data analysis to spectral data analysis. Select your single stain controls, parameters to unmix, and build histograms which will automatically set gates on positive an negative populations. Choose to calculate the matrix and get off and running with your analysis and reporting. The results will live-update whenever a change is made to the Unmixing setup.

Watch the short video or full length webinars below to learn how to create a spectral unmixing matrix

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Manual and Tutorials

Spectral Unmixing in the FCS Express Manual

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