Spectrum Analysis

Spectral flow cytometry is used to sample more parameters per single cell or particle than traditional flow cytometry. In contrast to conventional flow cytometers, spectral flow analyzers and sorters substitute mirrors, optical filters, and photomultiplier tubes (PMTs) with a spectrograph and multichannel detector to use fluorescence or Raman spectroscopy.

 

FCS Express has the capability to read and display raw spectral data from Cytek and Sony instruments through Spectrum Plots.  Spectral data plotted in Spectrum plots links directly the standard flow cytometry single cell measurements to allow seamless transitions between standard flow cytometry analysis and spectral cytometry analysis.

 

Supported Data Formats:

 

Sony Instruments:
oSpectral data is stored with the .SRAW file format with both an .SRAW and .FCS file being produced. To analyze data from Sony instruments in spectrum plots the .sraw and .fcs files produced by the instrument must be placed in the same source folder. The names of the .SRAW and .FCS files must match.

 

 

Cytek Instruments:
oSpectral data is stored with the standard .FCS file format however two files will be produced, an "unmixed" data file and a "mixed" data file. The unmixed data file will have the same name as the mixed data file except it will use a suffix of "Unmixed" in the data file name. For instance:

 

 sample1.fcs and sample1_Unmixed.fcs

 

The file containing the suffix of "Unmixed" contains the FCS file data that was unmixed via the Cytek system while the file with the standard file name remains mixed.
To view spectrum plots for the unmixed data set both data files most be placed in the same source folder with the names matching except for the "Unmixed" suffix.
To view spectrum plots for the mixed data set, only the original file without the "Unmixed" suffix must be present.

 

Custom Format for "Virtual Spectral Data":
oFCS Express allows you to visualize any .FCS data files as a "Virtual Spectral" data file via the addition of special keywords to the data set. A virtual spectra parameter may be created by indicating the original parameter channels to use in a spectral plot. For instance, you may choose to include FL1, FL2, FL3, FL4, etc as the parameters used to create a virtual spectral parameter for the data set and then use all of the standard Spectrum Plot functionality in FCS Express for further exploration and analysis of the data set.

 

An Extra Keywords file may be created to include all of the necessary keywords to produce a virtual spectral data file.

 

Keywords necessary for creating virtual spectral data:

 

 Download Example Virtual Spectral Data extra keywords file

 

 

Keyword

Explanation

Example

FCSE_SPECTRA1NAME=

Required: Indicates the name of the new spectral parameter.

FCSE_SPECTRA1NAME=FL

FCSE_SPECTRA1PARAMETERS=

Required:Indicates which of the original parameters to use to create a new virtual spectral parameter. Parameter names must match your user preferences for Keyword To Use as Parameter Name

FCSE_SPECTRA1PARAMETERS=FL1-H,FL2-H,FL3-H

FCSE_SPECTRA1LABELS=

Required:Indicates the labels to be used for each channel in Spectrum Plots.

FCSE_SPECTRA1LABELS=One,Two,Three

FCSE_COLUMNTODELETE_STRICT=

Optional: Comma separated exact list of parameters to delete after the import process. An error will be given if a parameter is not found for deletion. Parameter names must match your user preferences for Keyword To Use as Parameter Name

FCSE_COLUMNTODELETE_STRICT=FL1-H,FL2

FCSE_COLUMNTODELETE=

Optional: Comma separated list of parameters to delete after the import process. Any parameters not found will be ignored. Parameter names must match your user preferences for Keyword To Use as Parameter Name

FCSE_COLUMNTODELETE=FL1-H,FL2-H

 

 

Any example of creating a virtual spectral file for an original file containing FITC-A, PerCP-Cy5-5-A, PE-Cy7-A, APC-A, APC-Cy7-a is shown below. The original FCS file is shown on the right in a density plot and the resulting virtual spectral file for events in Gate 1 shown in a spectrum plot is on the left.

virtualspectraexample