Cytometry and Antibody Technology

Spectral Unmixing

Spectral Flow Cytometry

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Spectral unmixing is a mathematical algorithm that distinguishes the multiple fluorophore signatures within a multicolor tube and puts the each signature into its own parameter. This resources page contains a variety of information to better understand spectral unmixing and the best practices regarding spectral unmixing in a full spectrum flow cytometry experiment.

Background Information

Basics of Performing Spectral Unmixing in SpectroFlo

Many Aurora users prefer to perform spectral unmixing in the SpectroFlo application. After unmixing is correct and complete, you can complete your analysis in any other analysis application, such as FlowJo or FCS Express. These two resources provide the best practices for properly setting up the unmixing wizard in SpectroFlo.

The Right and Wrong Way to Set Up Automated Compensation Tools: How to Achieve Accurate Compensation

Note: This post applies to spectral unmixing as well.

When calculating compensation, automated tools are the gold standard. However, people often struggle to get good results from the automated compensation tools and will turn to manual compensation to fix any errors. Why is it difficult to get accurate results…

Download Slides: Performing Spectral Unmixing – 07-2021(PDF)

Advanced Unmixing Tips

Depending on the experiment setup, special considerations may be needed. These resources provide additional information to consider when performing a titration experiment, when deciding if beads or cells controls are optimal for unmixing, or when the experiment contains multiple unstained controls.


Autofluorescence is a hot topic in flow cytometery and researchers are continually learning more about it. This podcast highlights some points that must be considered when factoring autofluorescence into a spectral flow cytometry experiment, but doesn’t provide any detailed tutorials.

Fixing Unmixing Errors

Are you finding unmixing errors in your data? This blog post will provide a basic workflow for determining the cause of unmixing errors.

How to Fix Compensation Errors (and Unmixing Errors) – Bad Data Part 4

I have an unpopular opinion: I love compensation. Usually when I bring up compensation I’m met with a chorus of frustrated noises. But to me compensation is an opportunity to solve a complicated puzzle – and I really love a good puzzle. In this post I’m going to…