Spectral variants

Let each cell use the version of each fluorophore's spectrum that fits it best, to reduce spillover spread and skews between similar dyes.

A fluorophore’s spectrum is not quite the same in every cell: tandem dyes break down to different degrees, and a dye’s local environment shifts its emission a little. The Spectral Variants page finds that variation in each single-stained control. Unmixing can then choose, for every cell, the variant of each fluorophore that fits it best, together with the cell’s autofluorescence. This is the per-cell optimisation of the AutoSpectral package.

It needs AF profiles with at least two spectra assigned to your samples (Autofluorescence). It is an alternative to the noise model; you cannot use both.

Compute and use the variants

Press Compute Spectral Variants.
- The variants of each fluorophore are found by clustering the background-corrected, scatter-matched positive events of its single-stained control.
- Near-identical variants are merged, keeping at most 10 per fluorophore by default (Advanced Settings).
- A necessity score estimates how much each fluorophore’s variation would change the unmixing of the rest of the panel. Only fluorophores above the threshold are optimised per cell; the others keep their reference spectrum.
- A fluorophore with too little clean signal keeps its single reference spectrum. This is not an error, just nothing to optimise.
Plot Spectral Variants shows the variants of a chosen fluorophore (faint) against its reference spectrum (solid).
Tick Use spectral variants for the experiment’s unmixing. This is not available while the noise model is in use.

Plot Spectral Variants for PE-Fire 810: the variant spectra in red around the reference spectrum in blue — Plot Spectral Variants for PE-Fire 810 in OMIP-102 (Konecny et al. 2024): the variants (red) around the reference spectrum (blue). They differ mostly in the PE detectors on the blue and yellow-green lasers, where tandem breakdown shows.

The comparison shows the current sample with per-cell AF alone, and with per-cell AF plus spectral variants. It is subsampled for speed.

What good looks like: the trumpet-shaped spread of a bright dye into its spectral neighbours narrows, and skews between similar dyes flatten, while the populations themselves stay where they were.

Settings and speed

Tick Show Advanced Settings for the settings of the variant search, the necessity score, the unmixing and the comparison. The unmixing settings are stored with the experiment.

As with the noise model:

the Unmixed Data tab shows per-cell AF first and swaps in the result when it is ready;
results are cached;
unmixed exports with spectral variants start with a time estimate.

While another long job runs, Compute Spectral Variants, the Use tick box and the unmixing settings are greyed out; see Long jobs.

Spectral variants

Compute and use the variants

Settings and speed

Further reading