Spectral Unmixing; Understanding Full Spectrum Flow Cytometry; Reference Spectra - Cytek Biosciences Aurora User Manual

Spectral Unmixing

Spectral unmixing is an important concept to understand how data is generated and analyzed
using the Aurora flow cytometer with SpectroFlo software. Spectral unmixing is used to identify the
fluorescence signal for each fluorophore used in a given experiment.

Understanding Full Spectrum Flow Cytometry

Because fluorophores emit light over a range of wavelengths, optical filters are typically used to
limit the range of frequencies measured by a given detector. However, when two or more
fluorophores are used, the overlap in wavelength ranges often makes it impossible for optical
filters to isolate light from a given fluorophore. As a result, light emitted from one fluorophore
appears in a non-primary detector (a detector intended for another fluorophore). This is referred to
as spillover. In conventional flow cytometry spillover can be corrected by using a mathematical
calculation called compensation. Single-stained controls must be acquired to calculate the amount
of spillover into each of the non-primary detectors.
The Aurora's ability to measure a fluorochrome's full emission spectra allows the system to use a
different method for isolating the desired signal from the unwanted signal. The key to differentiate
the various fluorochromes is for those to have distinct patterns or signatures across the full
spectrum. Because the system is looking at the full range of emission of a given fluorochrome, and
not only the peak emission, two dyes with similar emission but different spectral signatures can be
distinguished from each other. The mathematical method to differentiate the signals from multiple
fluorochromes is call spectral unmixing. Just as for compensation, single-stained controls,
identified in SpectroFlo software as Reference Controls, are still necessary, as they provide the full
fluorescence spectra information needed to perform spectral unmixing.
Spectrum plots from conventional spectrum viewer shows heavy overlap between Qdot 705 and BV711.
Spectrum plots from Aurora show distinct signatures for Qdot 705 and BV711.

Reference Spectra

Reference Controls, obtained by running single-stained and unstained samples, provide the
individual fluorescence spectra necessary to unmix the data. Either beads or cells can be stained
for use as Reference Controls. These controls can be acquired in the Reference Group of the
experiment during acquisition, or they can be acquired as Reference Controls in the QC & Setup
workspace. If Reference Controls are acquired in the QC & Setup workspace, they are stored and
can be used as Reference Controls for subsequent experiments.
16 Aurora User's Guide