Unmix FlowCode FCS Data

This function performs spectral unmixing on FCS data from FlowCode samples.

Usage

unmix.flowcode.fcs(
  fcs.file,
  spectra,
  asp,
  flow.control,
  af.spectra,
  spectra.variants,
  flowcode.spectra,
  thresholds.file = NULL,
  weights = NULL,
  cell.weighting = FALSE,
  cell.weight.regularize = TRUE,
  k = 10,
  output.dir = NULL,
  file.suffix = NULL,
  include.imaging = FALSE,
  parallel = TRUE,
  threads = if (parallel) 0 else 1,
  verbose = TRUE
)

Arguments

fcs.file

A character string specifying the path to the FCS file.

spectra

A matrix containing the spectral data.

asp

The AutoSpectral parameter list. Prepare using get.autospectral.param

flow.control

A list containing flow cytometry control parameters.

af.spectra

Spectral signatures of autofluorescences, normalized between 0 and 1, with fluorophores in rows and detectors in columns. Prepare using get.af.spectra. Required for FlowCodeUnmixe unmixing.

spectra.variants

Named list (names are fluorophores) carrying matrices of spectral signature variations for each fluorophore. Prepare using get.spectral.variants.

flowcode.spectra

Structured output from get.flowcode.spectra(), which details the combination-level spectral unmixing errors due to FRET-like artefacts.

thresholds.file

Optional, file name and path to the thresholds CSV file produced using the threshold setting app. Thresholds are provided by default as part of flowcode.spectra, but those will have been selected on the FlowCode backbone sample. If the thresholds for positivity of any of the fluorophores used to identify the FlowCodes differs in the fully stained samples being unmixed here, provide a new set of thresholds from the ThresholdApp. Run this on an unmixed sample (OLS from the instrument is fine), and refer to the new thresholds file here. Default is NULL, which will be ignored.

weights

Optional numeric vector of weights (one per fluorescent detector). Default is NULL, in which case weighting will be done by channel means (Poisson variance).

cell.weighting

Logical, whether to use cell-specific weighting for a more Poisson-like unmixing. Default is FALSE.

cell.weight.regularize

Logical, whether to regularize cell-specific weights towards the bulk mean weighting set by weights. 50:50 averaging. Default is TRUE. Only active if cell.weighting=TRUE.

k

Numeric, controls the number of variants tested for each fluorophore, autofluorescence and FRET spectrum. Default is 10. Values up to 10 provide additional benefit in unmixing quality, 1 will be fastest.

output.dir

A character string specifying the directory to save the unmixed FCS file. Default is NULL, which reverts to asp$unmixed.fcs.dir.

file.suffix

A character string to append to the output file name. Default is NULL.

include.imaging

A logical value indicating whether to include imaging parameters in the written FCS file. Default is FALSE.

parallel

Logical, default is TRUE, which enables parallel processing for per-cell unmixing methods.

threads

Numeric, defaults to all available cores if parallel=TRUE.

verbose

Logical, whether to send messages to the console. Default is TRUE.

Value

None. The function writes the unmixed FCS data to a file.