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. Cytek and Sony spectral data sets have special preferences for loading data sets that are managed via Spectral Data Instrument Specific Settings.

 

Unmixing of data sets from spectral instruments is also supported by FCS Express.

 

Supported Data Formats:

 

•Cytek Instruments, SpectroFlo® Version 1.0:

oSpectral data are 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 must 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.

 

 

•Cytek Instruments, SpectroFlow Version 2.0:

oSpectral data are stored with the standard .FCS file format; however, two files will be produced, an "unmixed" data file and a "raw" data file, which will have the identical names but saved to different data folders, "Unmixed" and "Raw," respectively, when exported from SpectroFlo®. The folders must be kept with the same relative paths (i.e. within the same parent folder).

 

▪To view spectrum plots for the unmixed data sets, both raw and unmixed data files must be present in their respective source folders with identical .FCS file names. For instance:

 

 Experiment folder

         Raw folder > sample1.fcs

         Unmixed folder > sample1.fcs

.

▪To view spectrum plots for the mixed data set, only the raw, or mixed, file must be present.  

 

 

 

•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.

 

 

 

•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 or more easily via use of the Merge to Spectra pipeline step.  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 and the Merge to Spectra pipeline step may facilitate easy creation of the needed file.

 

▪Keywords necessary for creating virtual spectral data:

 

Please note the necessary keywords can be automatically created using the Merge to Spectra Pipeline step

 

 Download Example Virtual Spectral Data extra keywords file

 

 

Keyword

Explanation

Examples

FCSE_SPECTRA1NAME=

Required: Indicates the name of the new spectral parameter.  The value of this keyword is up to the discretion of the user.

FCSE_SPECTRA1NAME=FL

 

or

 

FCSE_SPECTRA1NAME=Virtual Spectra

FCSE_SPECTRA1PARAMETERS=

Required: Indicates which parameters to use to create the virtual spectral parameter.

 

Parameters can be listed using one of the following notations:

•Parameter Name (i.e. the $PnN keyword)

•The label defined by your user preferences for Keyword for Parameter Matching.

 

Mixing the two notation (i.e. using the $PnN for some parameters and the label defined by your user preferences for Keyword for Parameter Matching for some other parameters) is also possible.

 

For each entry in the list FCS Express first tries to match the $PnN keywords and, if the match does not occur for one or more entries, FCS Express tries to match the label defined in the user preferences for Keyword for Parameter Matching.

 

It is suggested to list all parameters using the $PnN since this will speed up the virtual spectra creation, especially for data with a lot of parameters.

 

 

Regular Expressions (RegEx) can also be used to define multiple parameters in an easier way. The regular expression must be entered using the RegEx:<regular expression here> formula (please see examples at the right).

To learn more on Regular Expression, please refer to https://en.wikipedia.org/wiki/Regular_expression .

 

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

 

or

 

FCSE_SPECTRA1PARAMETERS=FITC-A,PE-A,PerCP-A,APC-A

 

or

 

FCSE_SPECTRA1PARAMETERS=V445-H,V530-H,V586-H,V615-H,V660-H

 

or

 

 

FCSE_SPECTRA1PARAMETERS=RegEx:<^FL>,SSC-A

The regular expression matches all the parameters starting with FL. The SSC-A directly select the SSC-A parameter. The spectra will be made of all the FL parameters and the SSC-A parameter.

 

 

 

 

FCSE_SPECTRA1LABELS=

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

 

For example, the value for this keyword can be sequential numbers or simply the same string of text that was entered for FCSE_SPECTRA1PARAMETERS.

FCSE_SPECTRA1LABELS=One,Two,Three

 

or

 

FCSE_SPECTRA1LABELS=FITC-A,PE-A,PerCP-A,APC-A

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 for Parameter Matching.

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 (Figure 5.57).

 

Figure 5.57 Example of virtual spectra data file. 

Figure 5.57 Example of virtual spectra data file. 

 

 

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