Introduction to Spectral Flow Cytometry

Spectral flow cytometry

Signals from all detected channels are used, regardless of the number of fluorochromes analyzed. The fluorescence intensity is additive, and sums up wherever the spectra overlap.

Conventional flow cytometry

Fluorescence is only measured in a few defined ranges along the spectrum, with one detector for each fluorochrome. Any fluorescence signal outside the defined detection ranges is not collected.

There is no signal subtraction. Instead, fluorescence from each cell is measured at many points throughout the spectrum to define the overall fluorescence.

This spectrum includes the sum of the fluorescence from all of the fluorochromes, as well as autofluorescence. The spectral data is the fluorescence intensity measured by each of the detectors for each and every cell. The intensity of each fluorochrome in each cell is then calculated based on a weighted least squares (WLS) method, and this parametric data is used to create plots and gates to analyze results.

The fluorescence measured in some detectors includes spillover from other fluorochromes. This spillover needs to be subtracted by the process of compensation.

In spectral cell analyzers, each fluorochrome uses all the detection channels to produce a spectral emission signal. This signal is converted into a reference spectrum for each dye, which can then be used in spectral unmixing calculations. Using a WLS-based algorithm, spectral unmixing takes into account the actual noise detected in a sample to ensure accurate estimation of each dye’s intensity, and allows auto-fluorescence to be handled as a separate color.

Conventional flow cytometry relies on compensation adjustments based on a spillover matrix approach. When the fluorescent signal from a single fluorochrome (Dye 1) is acquired, the signal intensity detected in FL1 is considered as the ‘signal’, and any signals due to Dye 1 in other channels are described as spillover or ‘photon spillover’. Signals from all other channels therefore make up the ‘spillover matrix’ that is used in compensation calculations. Unfortunately, this approach does not adequately manage residual noise that can directly affect dye intensity. In multi-parameter experiments, manual adjustment based on the spillover matrix can be subjective and dependent on individual user expertise. In addition, autofluorescence is not considered in this approach.