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Deconvolve Autofluorescence Background

Source: R/deconvolve_af_background.R
deconvolve.af.background.Rd

Removes autofluorescence from raw detector data by fitting it jointly with the panel rather than subtracting it beforehand. Each event is unmixed against a design of the autofluorescence basis stacked on the fluorophore spectra and only the fitted autofluorescence part is subtracted, so a fluorophore's abundance and the background amount are estimated together instead of competing for the same signal.

Fluorophores whose spectra lie close to the span of the autofluorescence basis cannot be separated from it, and projecting them out removes real signal. The returned hotspot matrix reports that coupling. When target is supplied, components coupling to that fluorophore above max.hotspot are dropped from its fit; the leading component is always retained, because every event carries mean background.

Usage

deconvolve.af.background(
  raw.data,
  spectra,
  af.basis,
  af.name = "AF",
  target = NULL,
  max.hotspot = 5,
  nonneg = TRUE
)

Arguments

raw.data

Numeric matrix (events x detectors), raw detector data.

spectra

Numeric matrix (fluorophores x detectors), reference spectra. Any autofluorescence row named by af.name is removed before fitting, since the basis supersedes it.

af.basis

Numeric matrix (components x detectors) from get.af.basis().

af.name

Character or NULL, the name of an autofluorescence row in spectra to drop. Default "AF".

target

Character or NULL. When supplied, autofluorescence components that this fluorophore cannot be separated from are dropped before fitting. Default NULL.

max.hotspot

Numeric, the hotspot scale above which a component is considered inseparable from target. Default 5.

nonneg

Logical, whether to clamp fitted autofluorescence coefficients at zero before subtraction, so background can only be removed and never added. Default TRUE.

Value

A named list:

residual

raw.data with the fitted autofluorescence removed.

background

The fitted autofluorescence contribution.

abundance

Fluorophore abundances from the joint fit.

af.coef

Per-event autofluorescence component coefficients, clamped at zero when nonneg is TRUE.

af.coef.raw

The same coefficients before clamping, for use as regressors.

hotspot

Hotspot matrix of the joint design.

af.used, af.dropped

Component names kept and dropped.