The FCS File Options→Instrument Specific Settings category (Figure 20.52 (This option only appears if you are using FCS Express Flow or FCS Express Plus.) ) allows the user to set default preferences based on specific settings associated with specific date file types and instruments. Identification rules, generally based on a keyword stored in and recognized from the data file, trigger FCS Express to use a specific set of preferences when visualizing data sets on plots.

 

Instrument specific settings control the Keyword to use as the parameter name on plot axis, the keyword to use for parameter matching, axis range, scaling, and division factors for scatter and fluorescence parameters, and Merged FCS File Import Options. They also control settings associated with spectral data sets from Cytek and Sony instruments. A set of default Instrument Specific Settings is provided with FCS Express for instruments that generally require customizations to preferences to improve the display of data so it matches data displays from the acquisition software. Additional settings may be added at any time and the default settings may be adjusted or disabled if required.

 

Instrument Specific Settings will first try to identify if the data set loaded matches any identification rules part of any specific settings. FCS Express will attempt to match and give priority to the instrument specific settings based on the order of settings in the Instrument Specific Settings list. As an example, data from a FACScan instrument may be identified by both the Default FCS 2.0 and FACScan settings.  If the FACScan setting is higher in the Instrument Specific Settings list than the Default FCS 2.0 setting, it will be applied and vice versa.

 

If no matches are found, the Default FCS 2.0 or Default FCS 3.0 settings will be used. Please note that the default FCS 2.0 and FCS 3.0 Instrument Name and Identification Rules are the only ones that may not be modified, however, the individual settings as part of these defaults may be changed.

 

Figure 20.53   FCS File Options> Instrument Specific Settings dialog (top portion). Settings show are explained in table below. 

Figure 20.53   FCS File Options> Instrument Specific Settings dialog (top portion). Settings show are explained in table below. 

 

Instrument Specific Settings dialog options:

Option

Explanation

Restore Default Settings

Restores the list of Instruments and Instrument Specific Settings back to the default set and settings provided with FCS Express.

Duplicate

When an instrument specific settings is selected from the list, pressing Duplicate will create a copy of the setting. The copy maybe be renamed in the Instrument Name section and new identification rules and settings may be defined.

Add

The add button will allow you to create your own customized instrument specific setting from scratch. When the Add button is clicked a dropdown menu will appear allowing the choice of:

General
Cytek
Sony
Merged FCS Files

 

Choose from one of the options in the list to best match the data source type you intend to create an instrument specific settings for.

Remove

When an instrument specific settings is selected from the list, pressing Remove, will remove the setting from the list. Please note that Default FCS 2.0 and Default FCS 3.0 settings may not be removed.

Up and Down Arrows

When an instrument specific settings is selected from the list, pressing the Up or Down arrows will move the setting up or down the list. Settings higher in the list will be given priority over those lower in the list.

 

Instrument Specific Setting Options shared by all default settings and choices:

 

Option

Explanation

Instrument Name

Controls the name displayed in the Instrument Specific Settings list for the setting currently selected. Note this may not be changed for Default FCS 2.0 and Default FCS 3.0 settings.

Identification Rule

The instrument origin of data files will generally be recognized by the cytometer ($CYT) keyword, or a formula which evaluates multiple keywords, that are recorded as metadata by the cytometer at the time of acquisition.  Several Instrument-Specific Settings and Identification Rules have been included by default. Identification rules may be edited or added following the syntax with quotes (with $CYT as an example):  "<$CYT>" = "value of the $CYT keyword for your data files".  Operators such as OR, AND, NOT, and other common operators, may also be used. For instance '("<$CYT>" = "Accuri C6") or ("<$CYT>" = "BD Accuri C6")'

 

General Options:

Option

Explanation

Value to start for log parameters

Choose the starting value for parameters displayed in the log scale from the drop-down list.

Keyword To Use as Parameter Name

Choose a keyword (or combination of keywords) to use as parameter names from the drop-down list.

Keyword for Parameter Matching

Choose from Same as Display Name, Name, Stain, Name and Stain, or Stain and Name. When FCS Express attempts to match parameters, for instance, when defining compensation controls, these settings will be used to attempt matching.

 

Merge FCS File Import Options:

Option

Explanation

Merge FCS File Import Options

These options allow to automatically create the File Identifier (i.e. numeric) and/or the Classification (i.e. file names) Columns when FCS files are loaded as Merged FCS File (see the Adding Merged Files to the Data List and the Working with Heat Map chapter of the manual for more details on the Merge FCS File format).

An additional Classification keyword can also be added using any of the keywords available in the loaded files. If a keyword is specified and multiple files share the same value for that keyword, events from those files will be grouped together in the same well.

 

Scatter Parameters and Fluorescence Parameters Options:

Option

Explanation

Range Options

Default Range Options may be specified for Scatter and/or Fluorescence Parameters.  You may choose from four mutually exclusive options:

Automatic:  FCS Express will set the plot Range according to the Minimum and/or Maximum specified by the metadata (keywords) in the data file.  This is the default behavior of FCS Express, if no Instrument-Specific Settings are defined for the data files specified in Instrument Specific Settings > Condition.
Based on data:  Let FCS Express detect the actual minimum and maximum of your data for each parameter, and set the plot Range accordingly (a margin will be added so that data does not appear cut off on the plot boundaries).
Percentile:  FCS Express will set the plot Range according to the specified Percentile.  This is a convenient way to automatically not plot outliers which may located far beyond the data of interest.  In effect, your plots will already be zoomed into your highest-level region of interest.
Manual:  specify a Minimum and Maximum for the plot Range.

Scale Options

Default Scale Options may be specified for Scatter and/or Fluorescence Parameters. In the drop down menu, select the default scaling you wish to apply for the data files from the instrument specified in Instrument Specific Settings> Condition above.  The Automatic box will become unchecked once an option is specified.

Division Factor

The Division Factor may be specified for Scatter and/or Fluorescence Parameters.  In addition, the Data Scaling Factor may be specified for both types of parameters.  This allows for the data to be displayed with a different resolution (# of channels) than its native resolution.

 

Special Instrument Specific Settings for Spectral Data Sets

 

Cytek - SpectroFlo Specific Options

Option

Explanation

Import spectra as individual Parameters

When checked, Spectrum plots will allow the user to select any individual laser spectra and a merged spectra as a parameter, for instance, Violet, Blue, Red and Violet\Blue\Red will all be available. If unchecked, only one merged parameter for all laser spectra will be displayed, for instance, only one parameter for Violet\Blue\Red will be available.

Import spectra as Single Merged Parameter

When checked, a single merged parameter for all laser spectra will be available for selection. For instance, if Violet, Blue, and Red were collected, a new merged parameter for Violet\Blue\Red will be available. If unchecked, only individual spectra will be accessible as long as Import Spectra as individual Parameters is checked (above).

Spectra Parameter Label

The options allows users to define which Spectra Parameter to use as a label on spectrum plots, either Wavelengths or Parameter Names. If Wavelengths are selected, Spectrum plots will support displaying the numeric wavelength on the X axis, if Parameter Names is selected, Spectrum plots will support displaying the parameter names on the X axis, for instance, V1-A, V2-A, V3-A.

Delete fluorescence parameters after spectra merge

If checked, additional spectra parameters (for instance V1-A, V2-A, V3-A) will be removed when visualizing data in 2D and 1D plots. If unchecked, individual spectra parameters will be accessible in 2D and 1D plots at the end of the standard parameter list.

Merge Raw and Unmixed Cytek Files during import

When checked, if raw and unmixed data files exist in the appropriate file structure, and the unmixed data file is loaded into any plot(s), a resulting spectrum plot inserted for the data set will visualize all spectral parameters. If unchecked, when loading an unmixed data file, a spectrum plot for the data set will not be accessible since raw spectral data will not be merged with the unmixed results. When loading a raw data file, only scatter parameters will be accessible on 2D and 1D plots but the raw spectral data will be accessible in a spectrum plot.

Cytek File Merge Rule

Four options are available and described below:

Load User Selection: Uses the check box option set above for "Merge Raw and Unmixed Cytek Files during import"

Load both Raw and Unmixed Always: Loads both Raw and Unmixed into a merged data file for use in 2D, 1D, and spectrum plots regardless of the check box option set above for "Merge Raw and Unmixed Cytek Files during import"

Load Unmixed Only: Loads only the Unmixed version (no spectral parameters) of the data set when loading data.

Load Raw Only: Loads only the Raw version of the data set (only spectral parameters) when loading data.

 

 

 

Sony Specific Options

Option

Explanation

Spectrum Parameters to Read

The options allows users to define which Spectrum Parameters to Read, and to display on a Spectrum plot, either Wavelengths or Channels or both (one choice must be selected). If Wavelengths are selected, Spectrum plots will support displaying the wavelength on the X axis, if Channels is selected, Spectrum plots will support displaying the Channels on the X axis. If both are selected, Spectrum plots will support displaying both Wavelength and Channels on the X axis.