cytoflow.operations.autofluorescence

The cytoflow.operations.autofluorescence module contains two classes:

AutofluorescenceOp – corrects an Experiment for autofluorescence

AutofluorescenceDiagnosticView – a diagnostic to make sure that AutofluorescenceOp estimated its parameters correctly.

class cytoflow.operations.autofluorescence.AutofluorescenceOp

Bases: traits.has_traits.HasStrictTraits

Apply autofluorescence correction to a set of fluorescence channels.

The estimate function loads a separate FCS file (not part of the input Experiment) and computes the untransformed median and standard deviation of the blank cells. Then, apply subtracts the median from the experiment data.

To use, set the blank_file property to point to an FCS file with unstained or nonfluorescing cells in it; set the channels property to a list of channels to correct.

apply also adds the af_median and af_stdev metadata to the corrected channels, representing the median and standard deviation of the measured blank distributions.

channels

The channels to correct.

Type

List(Str)

blank_file

The filename of a file with “blank” cells (not fluorescent). Used to estimate the autofluorescence.

Type

File

blank_file_conditions

Occasionally, you’ll need to specify the experimental conditions that the blank tube was collected under (to apply the operations in the history.) Specify them here.

Type

Dict

Examples

Create a small experiment:

>>> import cytoflow as flow
>>> import_op = flow.ImportOp()
>>> import_op.tubes = [flow.Tube(file = "tasbe/rby.fcs")]
>>> ex = import_op.apply()

Create and parameterize the operation

>>> af_op = flow.AutofluorescenceOp()
>>> af_op.channels = ["Pacific Blue-A", "FITC-A", "PE-Tx-Red-YG-A"]
>>> af_op.blank_file = "tasbe/blank.fcs"

Estimate the model parameters

>>> af_op.estimate(ex)

Plot the diagnostic plot

>>> af_op.default_view().plot(ex)

Apply the operation to the experiment

>>> ex2 = af_op.apply(ex)

estimate(experiment, subset=None)

Estimate the autofluorescence from blank_file in channels specified in channels.

Parameters

• experiment (Experiment) – The experiment to which this operation is applied

• subset (Str (default = “”)) – An expression that specifies the events used to compute the autofluorescence

apply(experiment)

Applies the autofluorescence correction to channels in an experiment.

Parameters

experiment (Experiment) – the experiment to which this op is applied

Returns

a new experiment with the autofluorescence median subtracted. The corrected channels have the following metadata added to them:

• af_median : Float The median of the non-fluorescent distribution

• af_stdev : Float The standard deviation of the non-fluorescent distribution

Return type

Experiment

default_view(**kwargs)

Returns a diagnostic plot to see if the autofluorescence estimation is working.

Returns

An diagnostic view, call AutofluorescenceDiagnosticView.plot to see the diagnostic plots

Return type

IView

class cytoflow.operations.autofluorescence.AutofluorescenceDiagnosticView

Bases: traits.has_traits.HasStrictTraits

Plots a histogram of each channel, and its median in red. Serves as a diagnostic for the autofluorescence correction.

op

The AutofluorescenceOp whose parameters we’re viewing. Set automatically if you created the instance using AutofluorescenceOp.default_view.

Type

Instance(AutofluorescenceOp)

plot(experiment, **kwargs)

Plot a faceted histogram view of a channel